HDAC9 polypeptides and polynucleotides and uses thereof

ABSTRACT

The present invention features substantially pure HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), an HDRP(ΔNLS) polypeptides, and isolated nucleic acid molecules encoding those polypeptides. The present invention also features vectors containing HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) nucleic acid sequences, and cells containing those vectors.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/298,173 filed on Jun. 14, 2001, U.S. Provisional Application No.60/311,686 filed on Aug. 10, 2001, and U.S. Provisional Application No.60/316,995, filed on Sep. 4, 2001. The entire teachings of the aboveapplications are incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by grant CA-0974823from the National Cancer Institute. The Government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

The N-terminal tails of core histones are covalently modified bypost-translational modifications, including acetylation andphosphorylation. Evidence suggests that these covalent modificationsplay important roles in several biological activities involvingchromatin, e.g., transcription and replication. Histone deacetylases(HDACs) catalyze the removal of the acetyl group from the lysineresidues in the N-terminal tails of nucleosomal core histones resultingin a more compact chromatin structure, a configuration that is generallyassociated with repression of transcription.

Five proteins and/or open reading frames in yeast (RPD3, HDA1, HOS 1,HOS2 and HOS3) that share significant homology in the catalytic domainhave been identified as HDACs based upon their sequence homology tohuman HDAC1. To date, eight HDACs have been identified in mammaliancells, and classified into two classes based on their structure andsimilarity to yeast RPD3 or HDA1 proteins. Recently, Sir2 familyproteins that are structurally unrelated to the five proteinsaforementioned have been identified as NAD-dependent HDACs. Class IHDACs are the yeast RPD3 homologs HDAC1, 2, 3, and 8, and are composedprimarily of a catalytic domain. Class II HDACs are the yeast HDA1homologs HDAC4, 5, 6, and 7. HDAC4, 5, and 7 contain a longnon-catalytic N-terminal end and a C-terminal HDAC catalytic domainwhile HDAC6 has two HDAC catalytic domains.

It has also been determined that histone deacetylases can be sensitiveto small molecules, including trichostatin A (TSA), trapoxin, andbutyrate. For example, the yeast RPD3 and HDA1 and mammalian HDAC1, 2,3, 4, 5, 6, 7 and 8 are sensitive to inhibition by trichostatin A (TSA).The Sir2 family HDACs, yeast HOS3 and Drosophila melanogaster dHDAC6,however, appear to be relatively insensitive to TSA. A class of hybridbipolar compounds, such as suberoylanilide hydroxarmic acid (SAHA) havealso been shown to inhibit histone deacetylases and induce terminaldifferentiation and/or apoptosis in various transformed cells. Examplesof such compounds can be found in U.S. Pat. No. 5,369,108, issued onNov. 29, 1994, U.S. Pat. No. 5,700,811, issued on Dec. 23, 1997, andU.S. Pat. No. 5,773,474, issued on Jun. 30, 1998 to Breslow et al., aswell as U.S. Pat. No. 5,055,608, issued on Oct. 8, 1991, and U.S. Pat.No. 5,175,191, issued on Dec. 29, 1992 to Marks et al., the entirecontent of all of which are hereby incorporated by reference.

The identification of the mechanisms by which histones are deacetylated,and the characterization of histone deacetylase function would be ofgreat benefit in understanding how gene transcription is controlled, howthe cell cycle is regulated, and how cells are signaled to undergoterminal differentiation and/or apoptosis. Elucidation of suchmechanisms can lead to improved therapeutics for many diseases, inparticular those characterized by cell proliferation or a lack of celldifferentiation or apoptosis, for example, cancer.

SUMMARY OF THE INVENTION

The present invention relates to isolated or recombinant histonedeacetylase polypeptides, and isolated histone deacetylase nucleic acidmolecules encoding those polypeptides, as well as vectors and cellscontaining those isolated nucleic acid molecules.

In one aspect of the invention, the isolated or recombinant histonedeacetylase polypeptide is selected from a) an isolated or recombinantpolypeptide comprising SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, or SEQ ID NO: 10; and b) a polypeptide having at least 60%sequence identity with any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO: 8, or SEQ ID NO: 10. In one embodiment, the isolated orrecombinant histone deacetylase polypeptide consists of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. In anotherembodiment, the isolated or recombinant histone deacetylase polypeptideis mammalian; preferably, the isolated or recombinant histonedeacetylase polypeptide is human.

In another aspect, the invention features an isolated nucleic acidmolecule selected from a) an isolated nucleic acid comprising SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; b) acomplement of an isolated nucleic acid comprising SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; c) an isolatednucleic acid encoding a histone deacetylase polypeptide of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10; d) acomplement of an isolated nucleic acid encoding a histone deacetylasepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,or SEQ ID NO: 10; e) a nucleic acid that is hybridizeable under highstringency conditions to a nucleic acid molecule that encodes any of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, or a complementthereof; or f) a nucleic acid molecule that is hybridizeable under highstringency conditions to a nucleic acid comprising SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, or SEQ ID NO: 7; and g) an isolated nucleic acidmolecule that has at least 55% sequence identity with any one of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or acomplement thereof. In one embodiment, the isolated nucleic acidmolecule consists of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, or SEQ ID NO: 9. In another embodiment, the isolated nucleic acidmolecule is mammalian; preferably, the isolated nucleic acid molecule ishuman.

In other aspects, the invention features a vector comprising theisolated histone deacetylase nucleic acid molecule described above, acell comprising the vector, and a cell comprising the isolated histonedeacetylase nucleic acid molecule described above.

In another aspect, the invention features a purified antibody thatselectively binds a histone deacetylase polypeptide described above.

In yet another aspect, the invention features a method of identifying acompound that modulates expression of a histone deacetylase nucleic acidmolecule described above. The method comprises the steps of a)contacting the nucleic acid molecule with a candidate compound underconditions suitable for expression; and b) assessing the level ofexpression of the nucleic acid molecule. A candidate compound thatincreases or decreases expression of the nucleic acid molecule relativeto a control is a compound that modulates expression of the nucleic acidmolecule. In one embodiment, the method is carried out in a cell oranimal. In another embodiment, the method is carried out in a cell freesystem.

The invention also features a method of treating a cell proliferationdisease, an apoptotic disease, or a cell differentiation disease, forexample, cancers such as lymphoma, leukemia, melanoma, ovarian cancer,breast cancer, pancreatic cancer, prostate cancer, colon cancer, andlung cancer and myeloproliferative disorders, including polycythemiavera, essential thrombocythemia, agnogenic myeloid metaplasia, andchronic myelogenous leukemia in an individual, comprising administeringa compound identified by the above method.

In still another aspect, the invention features a method of identifyinga compound that modulates the enzymatic activity of the histonedeacetylase polypeptide described above. The method comprises the stepsof a) contacting the polypeptide with a candidate compound underconditions suitable for enzymatic reaction; and b) assessing theactivity level of the polypeptide. A candidate compound that increasesor decreases the activity level of the polypeptide relative to a controlis a compound that modulates the enzymatic activity of the polypeptide.In one embodiment, the method is carried out in a cell or animal. Inanother embodiment, the method is carried out in a cell free system.

In yet another embodiment, the polypeptide is further contacted with asubstrate for the polypeptide, wherein the substrate is selected fromthe group consisting of a cell proliferation disease binding agent, anapoptotic disease binding agent, and a cell differentiation diseasebinding agent. In one embodiment, the candidate compound is aninhibitor. In another embodiment, candidate compound is an activator.

In another aspect, the invention features a method of identifying acompound that modulates the transcriptional repression activity of thehistone deacetylase polypeptide described above. The method comprisesthe steps of a) contacting the polypeptide with a candidate compoundunder conditions suitable for a transcriptional repression reaction; andb) assessing the transcriptional repression activity level of thepolypeptide. A candidate compound that increases or decreases thetranscriptional repression activity level of the polypeptide relative toa control is a compound that modulates the transcriptional repressionactivity of the polypeptide. In one embodiment, the method is carriedout in a cell or animal. In another embodiment, the method is carriedout in a cell free system.

In yet another embodiment, the polypeptide is further contacted with asubstrate for the polypeptide, wherein the substrate is selected fromthe group consisting of a cell proliferation disease binding agent, anapoptotic disease binding agent, and a cell differentiation diseasebinding agent. In one embodiment, the candidate compound is aninhibitor. In another embodiment, candidate compound is an activator.

In another aspect, the invention features a method of identifying acompound that modulates expression of a histone deacetylase nucleic acidmolecule described above. The method comprises the steps of a) providinga nucleic acid molecule comprising a promoter region of the histonedeacetylase nucleic acid molecule described above, or part of such apromoter region, operably linked to a reporter gene; b) contacting thenucleic acid molecule or with a candidate compound; and c) assessing thelevel of the reporter gene. A candidate compound that increases ordecreases expression of the reporter gene relative to a control is acompound that modulates expression of the histone deacetylase nucleicacid molecule described above. In one embodiment, the method is carriedout in a cell.

In still another aspect, the invention features a method of identifyinga polypeptide that interacts with a histone deacetylase polypeptidedescribed above in a yeast two-hybrid system. The method comprises thesteps of a) providing a first nucleic acid vector comprising a nucleicacid molecule encoding a DNA binding domain and the histone deacetylasepolypeptide described above; b) providing a second nucleic acid vectorcomprising a nucleic acid encoding a transcription activation domain anda nucleic acid encoding a test polypeptide; c) contacting the firstnucleic acid vector with the second nucleic acid vector in a yeasttwo-hybrid system; and d) assessing transcriptional activation in theyeast two-hybrid system. An increase in transcriptional activationrelative to a control indicates that the test polypeptide is apolypeptide that interacts with the histone deacetylase polypeptidedescribed above.

The invention also features a pharmaceutical composition comprising ahistone deacetylase polypeptide described above.

In addition, the present invention features a method of diagnosing acell proliferation disease, an apoptotic disease, or a celldifferentiation disease in a subject. The method comprises the steps ofa) obtaining a sample from the subject; and b) assessing the level ofactivity or expression of the histone deacetylase polypeptide describedabove or the level of the nucleic acid molecule described above in thesample. If the level is increased relative to a control, then thesubject has an increased likelihood of having a cell proliferationdisease, an apoptotic disease, or a cell differentiation disease, and ifthe level is decreased relative to a control, then the subject has adecreased likelihood of having a cell proliferation disease, anapoptotic disease, or a cell differentiation disease. In one embodiment,the polypeptide level is assayed using immunohistochemistry techniques.In another embodiment, the nucleic acid molecule level is assayed usingin situ hybridization techniques.

Compounds and/or polypeptides identified in the above-describedscreening methods are also part of the present invention.

DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the order in which FIGS. 1A-1Oshould be viewed.

FIGS. 1A-1C show the cDNA sequence of HDAC9 (SEQ ID NO: 1). The arrowsand numbers in the HDAC9 sequence indicate exons. The boxed portion ofthe sequence indicates the HDAC domain.

FIGS. 1D-1G show the cDNA sequence of HDAC9a (SEQ ID NO: 3). The arrowsand numbers in the HDAC9a sequence indicate exons. The boxed portion ofthe sequence indicates the HDAC domain.

FIGS. 1H-1I show the cDNA sequence of HDRP(ΔNLS) (SEQ ID NO:9).

FIGS. 1J-1L show the cDNA sequence of HDAC9(ΔNLS) (SEQ ID NO:5).

FIGS. 1M-1O show the cDNA sequence of HDAC9a(ΔNLS) (SEQ ID NO:7).

FIG. 2 is a schematic representation of the order in which FIGS. 2A-2Eshould be viewed.

FIG. 2A shows the amino acid sequence of HDAC9 (SEQ ID NO: 2).

FIG. 2B shows the amino acid sequence of HDAC9a (SEQ ID NO: 4).

FIG. 2C shows the amino acid sequence of HDAC9(ΔNLS) (SEQ ID NO: 6).

FIG. 2D shows the amino acid sequence of HDAC9a(ΔNLS) (SEQ ID NO: 8).

FIG. 2E shows the amino acid sequence of and HDRP(ΔNLS) (SEQ ID NO: 10).

FIG. 3 is a schematic representation of the order in which FIGS. 3A-3Cshould be viewed.

FIGS. 3A-3C show an amino acid sequence alignment of HDRP (SEQ ID NO:11), HDAC9 (SEQ ID NO: 2), HDAC9a (SEQ ID NO: 4), and HDAC4 (SEQ ID NO:12) polypeptides. Amino acid sequences of HDAC9 (GenBank Accession:AY032737; SEQ ID NO: 2) and HDAC9a (GenBank Accession: AY032738; SEQ IDNO: 4) are aligned with HDRP (GenBank Accession: BAA34464; SEQ D NO: 11)and HDAC4 (GenBank Accession: NP_(—)006028; SEQ ID NO: 12). Theidentical residues in all proteins are boxed with solid lines. Thesimilar residues are boxed with dotted lines.

FIG. 4 shows a schematic representation of the human HDAC9 genestructure. The striped boxes represent exons present in isoforms HDRP,HDAC9a, and HDAC9. The lines represent introns. Broken lines are usedfor larger introns (with size in base pair on top). The 5′ untranslatedregion cDNA and coding region cDNA are represented here. Exons 1-12encode a non-catalytic domain of the polypeptides, and exons 14-21encode the histone deacetylase catalytic domain of the polypeptides,which provide the polypeptides with deacetylase activity.

FIG. 5 is a schematic representation of the order in which FIGS. 5A-5Dshould be viewed.

FIGS. 5A-5D show the nucleic acid sequence of HDAC9, containing allexons expressed in the various isoforms of HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), and HDRP(ΔNLS) of the present invention (SEQ ID NO:13).

FIG. 6A is a scanned imaged of a multiple human tissue Northern blotthat was probed to determine mRNA expression of HDAC9 using a cDNA probethat recognizes both HDAC9 and HDAC9a. The tissues examined are lane 1,heart; lane 2, brain; lane 3, placenta; lane 4, lung; lane 5, liver;lane 6, skeletal muscle; lane 7, kidney; and lane 8, pancreas. Positionsof the RNA size marker in kilobases (kb) are indicated to the left ofthe blot.

FIG. 6B is a scanned image of an electrophoretic gel showing the resultsof RT-PCR analyses of mRNA from the same tissues as examined in theNorthern blot of FIG. 6A to determine the distribution of HDAC9 andHDAC9a mRNA among these tissues. PCR products were resolved by agarosegel electrophoresis and visualized by ethidium bromide under UV light. A1-kb DNA ladder was run on both sides of the gel with the size (in kb)indicated on the left. On the right side, the expected products forHDAC9 and HDAC9a are indicated as 9 and 9a, respectively.

FIG. 7 is a graph of HDAC enzymatic activity of HDACanti-FLAG-immunoprecipitated proteins isolated from vector control,HDAC9-FLAG, and HDAC9a-FLAG transfected 293T cells, as measured influorescence units using FLUOR DE LYS™ as a substrate in the presence orabsence of 1 μM TSA. Results are shown as the mean of three independentassays. The inset is a scanned image of an anti-FLAG Western blotshowing the amount of proteins used in the assay. V, Vector control; 9,HDAC9-FLAG; and 9a, HDAC9a-FLAG.

FIG. 8 is a graph of HDAC enzymatic activity of HDACanti-FLAG-immunoprecipitated proteins isolated from vector control, andHDAC9a-FLAG (treated with 2 μM SAHA or left untreated) transfected 293Tcells, as measured by ³H-acetic acid released from ³H-histones in thepresence or absence of 2 μM SAHA. Vector control; HDAC9a, HDAC9a-FLAG;and HDAC9a+, HDAC9a-FLAG+SAHA.

FIG. 9A shows a scanned image of a Western blot of 293T whole celllysate and anti-FLAG immunoprecipitates from 293T cells transfected withvector, HDAC9-FLAG or HDAC9a-FLAG using antibodies against MEF2 andFLAG. Top panel, anti-MEF2 Western; bottom panel, anti-FLAG Western. L,293T whole cell lysate; V, vector control IP; 9, HDAC9-FLAG IP; 9a,HDAC9a-FLAG IP.

FIG. 9B is a graph showing the transcription level of p3XMEF2-Luc in thepresence or absence of pcDNA3 empty vector (−), pCMV-MEF2C, and/or avector encoding pFLAG-HDAC9 or pFLAG-HDAC9a. p3XMEF2-Luc (100 ng) andpRL-TK (5 ng) were transfected into 293T cells with pcDNA3 empty vector(−) or with pCMV-MEF2C (100 ng) (+) along with the indicated amount ofpFLAG-HDAC9 or pFLAG-HDAC9a. pFLAG empty vector was used to adjust theDNA to an equal amount in each transfection. The firefly luciferaseactivity was first normalized to the co-transfected Renilla luciferaseactivity and the value for MEF2C alone was then set as 1. Results areshown as the mean of three independent transfections±standard deviation.

FIG. 10 shows a schematic representation of the HDAC domains of humannon-Sir2 family HDACs and HDRP. The boxes represent histone deacetylase(HDAC) domains.

FIG. 11 is a schematic representation of the order in which FIGS.11A-11F should be viewed.

FIGS. 11A-11F show the nucleotide sequence of the vectorpFLAG-CMV-5b-HDAC9 (VR1) (SEQ ID NO: 14). Lowercase letters are vectorbackbone, uppercase letters are HDAC9 sequence. “Acc” was added at thebeginning of the HDAC9 sequence for translation initiation.

FIG. 12 is a schematic representation of the order in which FIGS. 12-1through 12-66 should be viewed.

FIGS. 12-1 through 12-66 show the nucleotide sequence of the vectorpFLAG-CMV-5b-HDAC9a (VR2), with restriction enzyme sites indicated (SEQID NO: 14).

FIG. 13 is a schematic representation of the order in which FIGS.13A-13E should be viewed.

FIGS. 13A-13E show the nucleotide sequence of the vectorpFLAG-CMV-5b-HDAC9a (VR2) (SEQ ID NO: 15). Lowercase letters are vectorbackbone, uppercase letters are HDAC9a sequence. “Acc” was added at thebeginning of the HDAC9a sequence for translation initiation.

FIG. 14 is a schematic representation of the order in which FIGS. 14-1through 14-61 should be viewed.

FIGS. 14-1 through 14-61 show the nucleotide sequence of the vectorpFLAG-CMV-5b-HDAC9a (VR2), with restriction enzyme sites indicated (SEQID NO: 15).

DETAILED DESCRIPTION OF THE INVENTION

A protein designated HDRP (See Zhou et al., Proc. Natl. Acad. Sci. USA,97:1056-1061 (2000)) (also called MITR (See Sparrow et al., EMBO J.18:5085-5098(1999); Zhang et al., J. Biol. Chem., 276:35-39 (2001); andZhang et al., Proc. Natl. Acad. Sci. USA, 98:7354-7359 (2001)) that is50% identical to the N-terminal domains of histone deacetylase 4 (HDAC4)and histone deacetylase 5 (HDAC5) was recently identified. The cloningand characterization of a novel histone deacetylase, HDAC9, of whichHDRP is an alternatively spliced isoform is described herein. The cDNAsequence of HDAC9 is shown in FIGS. 1A-1C (SEQ ID NO: 1), and the HDAC9amino acid sequence is shown in FIG. 2A (SEQ ID NO: 2). In addition tocloning HDAC9, other alternatively spliced isoforms of HDAC9, designatedas HDAC9a (a polypeptide that is 132 amino acids shorter at theC-terminal end than HDAC9), and isoforms of HDAC9, HDAC9a, and HDRPpolypeptides that lack the nuclear localization signal (NLS) in theN-terminal non-catalytic end of HDAC9, termed HDAC9(ΔNLS), HDAC9a(ΔNLS),and HDRP(ΔNLS), respectively were also identified. The cDNA sequence ofHDAC9a is shown in FIGS. 1D-1G (SEQ ID NO: 3), and the HDAC9a amino acidsequence is shown in FIG. 2B (SEQ ID NO: 4). The cDNA sequence of HDAC9lacking amino acids encoding an NLS (HDAC9(ΔNLS)) is shown in FIGS.1J-1L (SEQ ID NO: 5), and the HDAC9 lacking an NLS amino acid sequenceis shown in FIG. 2C (SEQ ID NO: 6). The cDNA sequence of HDAC9a encodinga polypeptide lacking an NLS (HDAC9a(ΔNLS)) is shown in FIGS. 1M-1O (SEQID NO: 7), and the HDAC9a lacking an NLS amino acid sequence is shown inFIG. 2D (SEQ ID NO: 8). The cDNA sequence of HDRP encoding a polypeptidelacking an NLS (HDRP(ΔNLS)) is shown in FIGS. 1H-1I (SEQ ID NO: 9), andthe HDRP lacking an NLS amino acid sequence is shown in FIG. 2E (SEQ IDNO: 10).

POLYPEPTIDES OF THE INVENTION

The present invention features isolated or recombinant HDAC9polypeptides, HDAC9a polypeptides, HDAC9(ΔNLS) polypeptides,HDAC9a(ΔNLS) polypeptides, and HDRP(ΔNLS) polypeptides, and fragments,derivatives, and variants thereof, as well as polypeptides encoded bynucleotide sequences described herein (e.g., other variants). As usedherein, the term “polypeptide” refers to a polymer of amino acids, andnot to a specific length; thus, peptides, oligopeptides, and proteinsare included within the definition of a polypeptide.

As used herein, a polypeptide is said to be “isolated,” “substantiallypure,” or “substantially pure and isolated” when it is substantiallyfree of cellular material, when it is isolated from recombinant ornon-recombinant cells, or free of chemical precursors or other chemicalswhen it is chemically synthesized. Typically, the HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide is isolated,substantially pure, or substantially pure and isolated when it has arelative increased concentration or activity of HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), in comparison to total HDACconcentration or activity. Preferably the increased activity orconcentration of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) is at least 2-fold, more preferably, at least 5-fold, andmost preferably, at least 10 fold, in comparison to total HDACconcentration or activity. In addition, a polypeptide can be joined toanother polypeptide with which it is not normally associated in a cell(e.g., in a “fusion protein”) and still be “isolated,” “substantiallypure,” or “substantially pure and isolated.” An isolated, substantiallypure, or substantially pure and isolated polypeptide may be obtained,for example, using affinity purification techniques described herein, aswell as other techniques described herein and known to those skilled inthe art.

By a “histone deacetylase polypeptide” is meant a polypeptide havinghistone deacetylase activity, transcription repression activity, and/orthe ability to deacetylate other substrates, for example, transcriptionfactors, including p53, CoRest, E2F, GATA-1, TFIIe, and TFIIF thatnormally have a nuclear or cytoplasmic location in a cell. A histonedeacetylase polypeptide is also a polypeptide whose activity can beinhibited by molecules having HDAC inhibitory activity. These moleculesfall into four general classes: 1) short-chain fatty acids (e.g.,4-phenylbutyrate and valproic acid); 2) hydroxamic acids(e.g. SAHA,Pyroxamide, trichostatin A (TSA), oxamflatin and CHAPs, such as, CHAP1and CHAP 31); 3) cyclic tetrapeptides (Trapoxin A, Apicidin andDepsipeptide (FK-228, also known as FR9011228); 4) benzamides (e.g.,MS-275); and other compounds such as Scriptaid. Examples of suchcompounds can be found in U.S. Pat. No. 5,369,108, issued on Nov. 29,1994, U.S. Pat. No. 5,700,811, issued on Dec. 23, 1997, and U.S. Pat.No. 5,773,474, issued on Jun. 30, 1998 to Breslow et al., U.S. Pat. No.5,055,608, issued on Oct. 8, 1991, and U.S. Pat. No. 5,175,191, issuedon Dec. 29, 1992 to Marks et al., as well as, Yoshida et al., Bioessays17, 423-430 (1995), Saito et al., PNAS USA 96, 4592-4597, (1999),Furamai et al., PNAS USA 98 (1), 87-92 (2001), Komatsu et al., CancerRes. 61(11), 4459-4466 (2001), Su et al., Cancer Res. 60, 3137-3142(2000), Lee et al., Cancer Res. 61(3), 931-934 and Suzuki et al. J. Med.Chem. 42(15), 3001-3003 (1999) the entire content of all of which arehereby incorporated by reference. Examples of such histone deacetylasepolypeptides include HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS),HDRP(ΔNLS); a substantially pure polypeptide comprising SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID xfxNO: 8, or SEQ ID NO: 10; and apolypeptide having preferably at least 60%, more preferably, 70%, 75%,80%, 85%, or 90%, and most preferably, 95% sequence identity to any oneof SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO:10, as determined using the BLAST program and parameters describedherein.

In one embodiment, the histone deacetylase polypeptide has histonedeacetylase activity, transcription repression activity, the ability todeacetylate substrates, or is inhibited by trichostatin A or a hybridpolar compound such as SAHA. In another embodiment, the HDAC9(ΔNLS)polypeptide has any two of the above biological activities. In stillanother embodiment, the HDAC9(ΔNLS) polypeptide has any three of theabove biological activities. In yet another embodiment, the HDAC9(ΔNLS)polypeptide has all of the above biological activities.

An HDAC9 polypeptide is a histone deacetylase polypeptide as describedabove. An HDAC9 polypeptide preferably has at least 60%, morepreferably, 70%, 75%, 80%, 85%, or 90%, and most preferably, 95%sequence identity to SEQ ID NO: 2, as determined using the BLAST programand parameters described herein. An HDAC9 polypeptide is also apolypeptide that comprises the amino acids encoded by exons 23, 24, 25and/or 26, and that does not comprise the amino acids encoded by exon 13of the HDAC9 nucleic acid sequence, as shown in FIGS. 1A-1C, FIG. 4, andFIGS. 5A-5D. Preferably, an HDAC9 polypeptide comprises the sequence ofSEQ ID NO: 2. More preferably, an HDAC9 polypeptide consists of thesequence of SEQ ID NO: 2. An HDAC polypeptide is also a polypeptidecomprising the amino acid sequence of the polypeptide encoded by thenucleic acid sequence of SEQ ID NO: 1.

An HDAC9a polypeptide is a histone deacetylase polypeptide as describedabove. An HDAC9a polypeptide preferably has at least 60%, morepreferably, 70%, 75%, 80%, 85%, or 90%, and most preferably, 95%sequence identity to SEQ ID NO: 4, as determined using the BLAST programand parameters described herein. An HDAC9a polypeptide is also apolypeptide that comprises the amino acids encoded by exon 22, and thatdoes not comprise the amino acids encoded by exons 13, 23, 24, 25, or 26of the HDAC9 nucleic acid sequence, as shown in FIGS. 1D-1G, FIG. 4, andFIGS. 5A-5D. Preferably, an HDAC9a polypeptide comprises the sequence ofSEQ ID NO: 4. More preferably, an HDAC9a polypeptide consists of thesequence of SEQ ID NO: 4. An HDAC9a polypeptide is also a polypeptidecomprising the amino acid sequence of the polypeptide encoded by thenucleic acid sequence of SEQ ID NO: 3.

An HDAC9(ΔNLS) is a histone deacetylase polypeptide as described above.An HDAC9(ΔNLS) polypeptide does not comprise a nuclear localizationsignal (NLS). An HDAC9(ΔNLS) polypeptide preferably has at least 60%,more preferably, 70%, 75%, 80%, 85%, or 90%, and most preferably, 95%sequence identity to SEQ ID NO: 6, as determined using the BLAST programand parameters described herein. An HDAC9(ΔNLS) polypeptide is also apolypeptide that comprises the amino acids encoded by exons 23, 24, 25,and/or 26, and that does not comprise the amino acids encoded by exons 7or 13 of the HDAC9 nucleic acid sequence, as shown in FIGS. 1J-1L, andFIGS. 5A-5D. Preferably, an HDAC9(ΔNLS) polypeptide comprises thesequence of SEQ ID NO: 6. More preferably, an HDAC9(ΔNLS) polypeptideconsists of the sequence of SEQ ID NO: 6. An HDAC9(ΔNLS) polypeptide isalso a polypeptide comprising the amino acid sequence of the polypeptideencoded by the nucleic acid sequence of SEQ ID NO: 5.

An HDAC9a(ΔNLS) polypeptide is a histone deacetylase polypeptide asdescribed above. An HDAC9a(ΔNLS) does not comprise a nuclearlocalization signal (NLS). An HDAC9a(ΔNLS) polypeptide preferably has atleast 60%, more preferably, 70%, 75%, 80%, 85%, or 90%, and mostpreferably, 95% sequence identity to SEQ ID NO: 8, as determined usingthe BLAST program and parameters described herein. An HDAC9a(ΔNLS)polypeptide is also a polypeptide that comprises the amino acids encodedby exon 22, and that does not comprise the amino acids encoded by exons7, 13, 23, 24, 25, or 26 of the HDAC9 nucleic acid sequence, as shown inFIGS. 1M-1O, and FIGS. 5A-5D. Preferably, an HDAC9a(ΔNLS) polypeptidecomprises the sequence of SEQ ID NO: 8. More preferably, an HDAC9a(ΔNLS)polypeptide consists of the sequence of SEQ ID NO: 8. An HDAC9a(ΔNLS)polypeptide is also a polypeptide comprising the amino acid sequence ofthe polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 7.

An HDRP(ΔNLS) polypeptide is a histone deacetylase polypeptide asdescribed above. An HDRP(ΔNLS) does not comprise a nuclear localizationsignal (NLS). An HDRP(ΔNLS) polypeptide preferably has at least 60%,more preferably, 70%, 75%, 80%, 85%, or 90%, and most preferably, 95%sequence identity to SEQ ID NO: 10, as determined using the BLASTprogram and parameters described herein. An HDRP(ΔNLS) polypeptide isalso a polypeptide that does not comprise the amino acids encoded byexons 7 or 13-26 of the HDAC9 nucleic acid sequence, as shown in FIGS.1H-1I and FIGS. 5A-5D. Preferably, an HDRP(ΔNLS) polypeptide comprisesthe sequence of SEQ ID NO: 10. More preferably, an HDRP(ΔNLS)polypeptide consists of the sequence of SEQ ID NO: 10. An HDRP(ΔNLS)polypeptide is also a polypeptide comprising the amino acid sequence ofthe polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 9.

The polypeptides of the invention can be purified to homogeneity. It isunderstood, however, that preparations in which the polypeptide is notpurified to homogeneity are useful. The critical feature is that thepreparation allows for the desired function of the polypeptide, even inthe presence of considerable amounts of other components. Thus, theinvention encompasses various degrees of purity. In one embodiment, thelanguage “substantially free of cellular material” includes preparationsof the polypeptide having less than about 30% (by dry weight) otherproteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins.

When a polypeptide is recombinantly produced, it can also besubstantially free of culture medium, i.e., culture medium representsless than about 20%, less than about 10%, or less than about 5% of thevolume of the polypeptide preparation. The language “substantially freeof chemical precursors or other chemicals” includes preparations of thepolypeptide in which it is separated from chemical precursors or otherchemicals that are involved in its synthesis. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of the polypeptide having less than about 30% (bydry weight) chemical precursors or other chemicals, less than about 20%chemical precursors or other chemicals, less than about 10% chemicalprecursors or other chemicals, or less than about 5% chemical precursorsor other chemicals.

In one embodiment, a polypeptide of the invention comprises an aminoacid sequence encoded by a nucleic acid molecule comprising a nucleotidesequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and complements andportions thereof, (e.g., a complement of any one of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or a portion of any oneof SEQ ID NO: 1, or SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ IDNO: 9).

The polypeptides of the invention also encompass fragments and sequencevariants. Variants include a substantially homologous polypeptideencoded by the same genetic locus in an organism, i.e., an allelicvariant, as well as other variants. Variants also encompass polypeptidesderived from other genetic loci in an organism, but having substantialhomology to a polypeptide encoded by a nucleic acid molecule comprisinga nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, andcomplements and portions thereof, or having substantial homology to apolypeptide encoded by a nucleic acid molecule comprising a nucleotidesequence selected from the group consisting of nucleotide sequencesencoding any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, or SEQ ID NO: 10. Variants also include polypeptides substantiallyhomologous or identical to these polypeptides but derived from anotherorganism, i.e., an ortholog. Variants also include polypeptides that aresubstantially homologous or identical to these polypeptides that areproduced by chemical synthesis. Variants also include polypeptides thatare substantially homologous or identical to these polypeptides that areproduced by recombinant methods.

As used herein, two polypeptides (or a region of the polypeptides) aresubstantially homologous or identical when the amino acid sequences areat least about 60-65%, typically at least about 70-75%, more typicallyat least about 80-85%, and most typically greater than about 90-95% ormore homologous or identical. A substantially identical or homologousamino acid sequence, according to the present invention, will be encodedby a nucleic acid molecule hybridizing to SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or a portion thereof, understringent conditions as more particularly described herein, or will beencoded by a nucleic acid molecule hybridizing to a nucleic acidsequence encoding SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, or portion thereof, under stringent conditions as moreparticularly described herein.

The percent identity of two nucleotide or amino acid sequences can bedetermined by aligning the sequences for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first sequence). Thenucleotides or amino acids at corresponding positions are then compared,and the percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences (i.e., %identity=# of identical positions/total # of positions×100). In certainembodiments, the length of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS),and HDRP(ΔNLS) amino acid or nucleotide sequence aligned for comparisonpurposes is at least 30%, preferably, at least 40%, more preferably, atleast 60%, and even more preferably, at least 70%, 80%, 90%, or 100% ofthe length of the reference sequence, for example, those sequencesprovided in FIGS. 1A-1O and 2A-2E. The actual comparison of the twosequences can be accomplished by well-known methods, for example, usinga mathematical algorithm. A preferred, non-limiting example of such amathematical algorithm is described in Karlin et al., Proc. Natl. Acad.Sci. USA, 90: 5873-5877 (1993). Such an algorithm is incorporated intothe BLASTN and BLASTX programs (version 2.2) as described in Schaffer etal., Nucleic Acids Res., 29: 2994-3005 (2001). When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., BLASTN) can be used. See http://www.ncbi.nlm.nih.gov, asavailable on Aug. 10, 2001. In one embodiment, the database searched isa non-redundant (NR) database, and parameters for sequence comparisoncan be set at: no filters; Expect value of 10; Word Size of 3; theMatrix is BLOSUM62; and Gap Costs have an Existence of 11 and anExtension of 1.

Another preferred, non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller, CABIOS (1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0), which is part of the GCG (Accelrys) sequencealignment software package. When utilizing the ALIGN program forcomparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12 , and a gap penalty of 4 can be used. Additionalalgorithms for sequence analysis are known in the art and includeADVANCE and ADAM as described in Torellis and Robotti, Comput. Appl.Biosci., 10: 3-5 (1994); and FASTA described in Pearson and Lipman,Proc. Natl. Acad. Sci USA, 85: 2444-8 (1988).

In another embodiment, the percent identity between two amino acidsequences can be accomplished using the GAP program in the GCG softwarepackage (available at http://www.accelrys.com, as available on Aug. 31,2001) using either a Blossom 63 matrix or a PAM250 matrix, and a gapweight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. In yetanother embodiment, the percent identity between two nucleic acidsequences can be accomplished using the GAP program in the GCG softwarepackage (available at http://www.cgc.com), using a gap weight of 50 anda length weight of 3.

The invention also encompasses HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9aΔNLS,and HDRP(ΔNLS) polypeptides having a lower degree of identity but havingsufficient similarity so as to perform one or more of the same functionsperformed by an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9aΔNLS, or HDRP(ΔNLS)polypeptide encoded by a nucleic acid molecule of the invention.Similarity is determined by conserved amino acid substitution. Suchsubstitutions are those that substitute a given amino acid in apolypeptide by another amino acid of like characteristics. Conservativesubstitutions are likely to be phenotypically silent. Typically seen asconservative substitutions are the replacements, one for another, amongthe aliphatic amino acids Ala, Val, Leu, and Ile; interchange of thehydroxyl residues Ser and Thr; exchange of the acidic residues Asp andGlu; substitution between the amide residues Asn and Gln; exchange ofthe basic residues Lys and Arg; and replacements among the aromaticresidues Phe and Tyr. Guidance concerning which amino acid changes arelikely to be phenotypically silent are found in Bowie et al., Science247: 1306-1310 (1990).

A variant polypeptide can differ in amino acid sequence by one or moresubstitutions, deletions, insertions, inversions, fusions, andtruncations or a combination of any of these. Further, variantpolypeptides can be fully functional or can lack function in one or moreactivities, for example, in histone deacetylase activity ortranscription repression activity. Fully functional variants typicallycontain only conservative variation or variation in non-criticalresidues or in non-critical regions. Functional variants can alsocontain substitution of similar amino acids that result in no change oran insignificant change in function. Alternatively, such substitutionsmay positively or negatively affect function to some degree.Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncations or a substitution, insertion, inversion, or deletion in acritical residue or critical region, such critical regions include theHDAC domains, which provide the polypeptide with deacetylase activity,as shown in the nucleic acid sequences of FIGS. 1A-1G, as well as in theschematic of FIG. 4.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science, 244: 1081-1085 (1989)). Thelatter procedure introduces a single alanine mutation at each of theresidues in the molecule (one mutation per molecule). The resultingmutant molecules are then tested for biological activity in vitro. Sitesthat are critical for polypeptide activity can also be determined bystructural analysis, such as crystallization, nuclear magneticresonance, or photoaffinity labeling (See Smith et al., J. Mol. Biol.,224: 899-904 (1992); and de Vos et al. Science, 255: 306-312 (1992)).

The invention also includes HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS),and HDRP(ΔNLS) polypeptide fragments of the polypeptides of theinvention. Fragments can be derived from a polypeptide comprising SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, orfrom a polypeptide encoded by a nucleic acid molecule comprising SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 or aportion thereof and the complements thereof or other variants. Thepresent invention also encompasses fragments of the variants of thepolypeptides described herein. Useful fragments include those thatretain one or more of the biological activities of the polypeptide aswell as fragments that can be used as an immunogen to generatepolypeptide-specific antibodies.

Biologically active fragments (peptides that are, for example, 6, 9, 12,15, 16, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100, or more amino acids inlength) can comprise a domain, segment, or motif, for example, an HDACdomain, that has been identified by analysis of the polypeptide sequenceusing well-known methods, e.g., signal peptides, extracellular domains,one or more transmembrane segments or loops, ligand binding regions,zinc finger domains, DNA binding domains, acylation sites, glycosylationsites, or phosphorylation sites.

Fragments can be discrete (not fused to other amino acids orpolypeptides) or can be within a larger polypeptide. Further, severalfragments can be comprised within a single larger polypeptide. In oneembodiment a fragment designed for expression in a host can haveheterologous pre- and pro-polypeptide regions fused to the aminoterminus of the polypeptide fragment and an additional region fused tothe carboxyl terminus of the fragment.

The invention thus provides chimeric or fusion polypeptides. Thesecomprise an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9aΔNLS, or HDRP(ΔNLS)polypeptide of the invention operatively linked to a heterologousprotein or polypeptide having an amino acid sequence not substantiallyhomologous to the polypeptide. “Operatively linked” indicates that thepolypeptide and the heterologous protein are fused in-frame. Theheterologous protein can be fused to the N-terminus or C-terminus of thepolypeptide. In one embodiment, the fusion polypeptide does not affectthe function of the polypeptide per se. For example, the fusionpolypeptide can be a GST-fusion polypeptide in which the polypeptidesequences are fused to the C-terminus of the GST sequences. Other typesof fusion polypeptides include, but are not limited to, enzymatic fusionpolypeptides, for example, P-galactosidase fusions, yeast two-hybrid GALfusions, poly-His fusions, and Ig fusions. Such fusion polypeptides,particularly poly-His fusions, can facilitate the purification ofrecombinant polypeptide. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a polypeptide can be increased byusing a heterologous signal sequence. Therefore, in another embodiment,the fusion polypeptide contains a heterologous signal sequence at itsN-terminus.

EP-A 0464 533 discloses fusion proteins comprising various portions ofimmunoglobulin constant regions. The Fc is useful in therapy anddiagnosis and thus results, for example, in improved pharmacokineticproperties (EP-A 0232 262). In drug discovery, for example, humanproteins have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists. (See Bennettet al., Journal of Molecular Recognition, 8: 52-58 (1995) and Johansonet al., The Journal of Biological Chemistry, 270,16: 9459-9471 (1995)).Thus, this invention also encompasses soluble fusion polypeptidescontaining a polypeptide of the invention and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclass (IgG, IgM, IgA, IgE).

A chimeric or fusion polypeptide can be produced by standard recombinantDNA techniques. For example, DNA fragments coding for the differentpolypeptide sequences are ligated together in-frame in accordance withconventional techniques. In another embodiment, the fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of nucleic acid fragmentscan be carried out using anchor primers that give rise to complementaryoverhangs between two consecutive nucleic acid fragments that cansubsequently be annealed and re-amplified to generate a chimeric nucleicacid sequence (see Ausubel et al., “Current Protocols in MolecularBiology,” John Wiley & Sons, (1998), the entire teachings of which areincorporated by reference herein). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTprotein). A nucleic acid molecule encoding a polypeptide of theinvention can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the polypeptide.

The substantially pure, isolated, or substantially pure and isolatedHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9aΔNLS, or HDRP(ΔNLS) polypeptide can bepurified from cells that naturally express it, purified from cells thathave been altered to express it (recombinant), or synthesized usingknown protein synthesis methods. In one embodiment, the polypeptide isproduced by recombinant DNA techniques. For example, a nucleic acidmolecule encoding the polypeptide is cloned into an expression vector,the expression vector introduced into a host cell, and the polypeptideexpressed in the host cell. The polypeptide can then be isolated fromthe cells by an appropriate purification scheme using standard proteinpurification techniques.

In general, HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9aΔNLS, and HDRP(ΔNLS)polypeptides of the present invention can be used as a molecular weightmarker on SDS-PAGE gels or on molecular sieve gel filtration columnsusing art-recognized methods. The polypeptides of the present inventioncan be used to raise antibodies or to elicit an immune response. Thepolypeptides can also be used as a reagent, e.g., a labeled reagent, inassays to quantitatively determine levels of the polypeptide or amolecule to which it binds (e.g., a receptor or a ligand) in biologicalfluids. The polypeptides can also be used as markers for cells ortissues in which the corresponding polypeptide is preferentiallyexpressed, either constitutively, during tissue differentiation, or in adiseased state. The polypeptides can be used to isolate a correspondingbinding agent, and to screen for peptide or small molecule antagonistsor agonists of the binding interaction. The polypeptides of the presentinvention can also be used as therapeutic agents.

NUCLEIC ACID MOLECULES OF THE INVENTION

The present invention also features isolated HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), and HDRP(ΔNLS) nucleic acid molecules.

By a “histone deacetylase nucleic acid molecule” is meant a nucleic acidmolecule that encodes a histone deacetylase polypeptide. Such histonenucleic acids include, for example, the HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule described in detailherein; an isolated nucleic acid comprising SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; a complement of an isolatednucleic acid comprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, or SEQ ID NO: 9; an isolated nucleic acid encoding a histonedeacetylase polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, or SEQ ID NO: 10; a complement of an isolated nucleic acidencoding a histone deacetylase polypeptide of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10; a nucleic acid that ishybridizeable under high stringency conditions to a nucleic acidmolecule that encodes any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,or SEQ ID NO: 8, or a complement thereof; a nucleic acid molecule thatis hybridizeable under high stringency conditions to a nucleic acidcomprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7;and an isolated nucleic acid molecule that has at least 55%, morepreferably, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, and most preferably,95% or 99% sequence identity with any one of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7,SEQID NO: 9, or a complement thereof.

An HDAC9 nucleic acid molecule is a nucleic acid molecule that encodesan HDAC9 polypeptide. In one embodiment, the HDAC9 nucleic acid moleculeis selected from: a nucleic acid molecule that comprises the nucleicacid sequence of SEQ ID NO: 1; a complement of an isolated nucleic acidcomprising SEQ ID NO: 1; an isolated nucleic acid encoding a histonedeacetylase polypeptide of SEQ ID NO: 2; a complement of an isolatednucleic acid encoding a histone deacetylase polypeptide of SEQ ID NO: 2;a nucleic acid that is hybridizeable under high stringency conditions toa nucleic acid molecule that encodes SEQ ID NO: 2; a nucleic acidmolecule that is hybridizeable under high stringency conditions to anucleic acid comprising SEQ ID NO: 1; and an isolated nucleic acidmolecule that has preferably, at least 55%, more preferably, 60%, 65%,70%, 75%, 80%, 85%, or 90%, and most preferably, 95% or 99% sequenceidentity with SEQ ID NO: 1, as determined using the BLAST program andparameters described herein. In another embodiment, the HDAC9 nucleicacid molecule consists of the nucleic acid sequence of SEQ ID NO: 1.

An HDAC9a nucleic acid molecule is a nucleic acid molecule that encodesan HDAC9a polypeptide. An HDAC9a nucleic acid molecule preferably has atleast 55%, sequence identity to SEQ ID NO: 3, In one embodiment, theHDAC9a nucleic acid molecule is selected from: a nucleic acid moleculethat comprises the nucleic acid sequence of SEQ ID NO: 3; a complementof an isolated nucleic acid comprising SEQ ID NO: 3; an isolated nucleicacid encoding a histone deacetylase polypeptide of SEQ ID NO: 4; acomplement of an isolated nucleic acid encoding a histone deacetylasepolypeptide of SEQ ID NO: 4; a nucleic acid that is hybridizeable underhigh stringency conditions to a nucleic acid molecule that encodes SEQID NO: 4; a nucleic acid molecule that is hybridizeable under highstringency conditions to a nucleic acid comprising SEQ ID NO: 3; and anisolated nucleic acid molecule that has preferably, at least 55%, morepreferably, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, and most preferably,95% or 99% sequence identity with SEQ ID NO: 3 or a complement thereof,as determined using the BLAST program and parameters described herein.In another embodiment, the HDAC9a nucleic acid molecule consists of thenucleic acid sequence of SEQ ID NO: 3.

An HDAC9(ΔNLS) nucleic acid molecule is a nucleic acid molecule thatencodes an HDAC9(ΔNLS) polypeptide. In one embodiment, the HDAC9(ΔNLS)nucleic acid molecule is selected from: a nucleic acid molecule thatcomprises the nucleic acid sequence of SEQ ID NO: 5; a complement of anisolated nucleic acid comprising SEQ ID NO: 5; an isolated nucleic acidencoding a histone deacetylase polypeptide of SEQ ID NO: 6; a complementof an isolated nucleic acid encoding a histone deacetylase polypeptideof SEQ ID NO: 6; a nucleic acid that is hybridizeable under highstringency conditions to a nucleic acid molecule that encodes SEQ ID NO:6; a nucleic acid molecule that is hybridizeable under high stringencyconditions to a nucleic acid comprising SEQ ID NO: 5; and an isolatednucleic acid molecule that has preferably, at least 55%, morepreferably, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, and most preferably,95% or 99% sequence identity with SEQ ID NO: 5 or a complement thereof,as determined using the BLAST program and parameters described herein.In another embodiment, the HDAC9(ΔNLS) nucleic acid molecule consists ofthe nucleic acid sequence of SEQ ID NO: 5.

An HDAC9a(ΔNLS) nucleic acid molecule is a nucleic acid molecule thatencodes an HDAC9a(ΔNLS) polypeptide. In one embodiment, the HDAC9a(ΔNLS)nucleic acid molecule is selected from: a nucleic acid molecule thatcomprises the nucleic acid sequence of SEQ ID NO: 7; a complement of anisolated nucleic acid comprising SEQ ID NO: 7; an isolated nucleic acidencoding a histone deacetylase polypeptide of SEQ ID NO: 8; a complementof an isolated nucleic acid encoding a histone deacetylase polypeptideof SEQ ID NO: 8; a nucleic acid that is hybridizeable under highstringency conditions to a nucleic acid molecule that encodes SEQ ID NO:8; a nucleic acid molecule that is hybrdizeable under high stringencyconditions to a nucleic acid comprising SEQ ID NO: 7; and an isolatednucleic acid molecule that has preferably, at least 55%, morepreferably, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, and most preferably,95% or 99% sequence identity with SEQ ID NO: 7 or a complement thereof,as determined using the BLAST program and parameters described herein.In another embodiment, the HDAC9a(ΔNLS) nucleic acid molecule consistsof the nucleic acid sequence of SEQ ID NO: 7.

An “HDRP(ΔNLS) nucleic acid molecule” is a nucleic acid molecule thatencodes an HDRP(ΔNLS) polypeptide. In one embodiment, the HDRP(ΔNLS)nucleic acid molecule is selected from: a nucleic acid molecule thatcomprises the nucleic acid sequence of SEQ ID NO: 9; a complement of anisolated nucleic acid comprising SEQ ID NO: 9; an isolated nucleic acidencoding a histone deacetylase polypeptide of SEQ ID NO: 10; acomplement of an isolated nucleic acid encoding a histone deacetylasepolypeptide of SEQ ID NO: 10; and an isolated nucleic acid molecule thathas preferably, at least 55%, more preferably, 60%, 65%, 70%, 75%, 80%,85%, or 90%, and most preferably, 95% or 99% sequence identity with SEQID NO: 9 or a complement thereof, as determined using the BLAST programand parameters described herein. In another embodiment, the HDRP(ΔNLS)nucleic acid molecule consists of the nucleic acid sequence of SEQ IDNO: 9.

The isolated nucleic acid molecules of the present invention can be RNA,for example, mRNA, or DNA, such as cDNA and genomic DNA. DNA moleculescan be double-stranded or single-stranded; single stranded RNA or DNAcan be either the coding, or sense, strand or the non-coding, orantisense, strand. The nucleic acid molecule can include all or aportion of the coding sequence of the gene and can further compriseadditional non-coding sequences such as introns and non-coding 3′ and 5′sequences (including regulatory sequences, for example). Additionally,the nucleic acid molecule can be fused to a marker sequence, forexample, a sequence that encodes a polypeptide to assist in isolation orpurification of the polypeptide. Such sequences include, but are notlimited to, those that encode a glutathione-S-transferase (GST) fusionprotein and those that encode a hemagglutinin A (HA) polypeptide markerfrom influenza.

An “isolated,” “substantially pure,” or “substantially pure andisolated” nucleic acid molecule, as used herein, is one that isseparated from nucleic acids that normally flank the gene or nucleotidesequence (as in genomic sequences) and/or has been completely orpartially purified from other transcribed sequences (e.g., as in an RNAor cDNA library). For example, an isolated nucleic acid of the inventionmay be substantially isolated with respect to the complex cellularmilieu in which it naturally occurs, or culture medium when produced byrecombinant techniques, or chemical precursors or other chemicals whenchemically synthesized. In some instances, the isolated material willform part of a composition (for example, a crude extract containingother substances), buffer system, or reagent mix. In othercircumstances, the material may be purified to essential homogeneity,for example, as determined by agarose gel electrophoresis or columnchromatography such as HPLC. Preferably, an isolated nucleic acidmolecule comprises at least about 50, 80, or 90% (on a molar basis) ofall macromolecular species present.

With regard to genomic DNA, the term “isolated” also can refer tonucleic acid molecules that are separated from the chromosome with whichthe genomic DNA is naturally associated. For example, the isolatednucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotides that flank the nucleic acidmolecule in the genomic DNA of the cell from which the nucleic acidmolecule is derived.

The HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acidmolecule can be fused to other coding or regulatory sequences and stillbe considered isolated. Thus, recombinant DNA contained in a vector isincluded in the definition of “isolated” as used herein. Also, isolatednucleic acid molecules include recombinant DNA molecules in heterologoushost cells, as well as partially or substantially purified DNA moleculesin solution. “Isolated” nucleic acid molecules also encompass in vivoand in vitro RNA transcripts of the DNA molecules of the presentinvention. An isolated nucleic acid molecule or nucleotide sequence caninclude a nucleic acid molecule or nucleotide sequence that issynthesized chemically or by recombinant means. Therefore, recombinantDNA contained in a vector are included in the definition of “isolated”as used herein.

Isolated nucleotide molecules also include recombinant DNA molecules inheterologous organisms, as well as partially or substantially purifiedDNA molecules in solution. In vivo and in vitro RNA transcripts of theDNA molecules of the present invention are also encompassed by“isolated” nucleotide sequences. Such isolated nucleotide sequences areuseful in the manufacture of the encoded polypeptide, as probes forisolating homologous sequences (e.g., from other mammalian species), forgene mapping (e.g., by in situ hybridization with chromosomes), or fordetecting expression of the gene in tissue (e.g., human tissue), such asby Northern blot analysis.

The present invention also pertains to variant HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) nucleic acid molecules thatare not necessarily found in nature but that encode an HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide. Thus, for example,DNA molecules that comprise a sequence that is different from thenaturally-occurring HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) nucleotide sequence but which, due to the degeneracy of thegenetic code, encode an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) polypeptide of the present invention are also the subject ofthis invention.

The invention also encompasses HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS),and HDRP(ΔNLS) nucleotide sequences encoding portions (fragments), orencoding variant polypeptides such as analogues or derivatives of anHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide.Such variants can be naturally-occurring, such as in the case of allelicvariation or single nucleotide polymorphisms, ornon-naturally-occurring, such as those induced by various mutagens andmutagenic processes. Intended variations include, but are not limitedto, addition, deletion, and substitution of one or more nucleotides thatcan result in conservative or non-conservative amino acid changes,including additions and deletions. Preferably, the HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleotide (and/or resultantamino acid) changes are silent or conserved; that is, they do not alterthe characteristics or activity of the HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide. In one preferred embodiment,the nucleotide sequences are fragments that comprise one or morepolymorphic microsatellite markers.

Other alterations of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) nucleic acid molecules of the invention can include, forexample, labeling, methylation, internucleotide modifications such asuncharged linkages (e.g., methyl phosphonates, phosphotriesters,phosphoamidates, and carbamates), charged linkages (e.g.,phosphorothioates or phosphorodithioates), pendent moieties (e.g.,polypeptides), intercalators (e.g., acridine or psoralen), chelators,alkylators, and modified linkages (e.g., alpha anomeric nucleic acids).Also included are synthetic molecules that mimic nucleic acid moleculesin the ability to bind to a designated sequences via hydrogen bondingand other chemical interactions. Such molecules include, for example,those in which peptide linkages substitute for phosphate linkages in thebackbone of the molecule.

The invention also pertains to HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS),and HDRP(ΔNLS) nucleic acid molecules that hybridize under highstringency hybridization conditions, such as for selectivehybridization, to a nucleotide sequence described herein (e.g., nucleicacid molecules that specifically hybridize to a nucleotide sequenceencoding polypeptides described herein, and, optionally, have anactivity of the polypeptide). In one embodiment, the invention includesvariants described herein that hybridize under high stringencyhybridization conditions (e.g., for selective hybridization) to anucleotide sequence comprising a nucleotide sequence selected from SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and thecomplement of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ D NO: 7, orSEQ ID NO: 9. In another embodiment, the invention includes variantsdescribed herein that hybridize under high stringency hybridizationconditions (e.g., for selective hybridization) to a nucleotide sequenceencoding an amino acid sequence of SEQ ID NO: 2 (HDAC9), SEQ ID NO: 4(HDAC9a), SEQ ID NO: 6 (HDAC9(ΔNLS)), SEQ ID NO: 8 (HDAC9a(ΔNLS)), orSEQ ID NO: 10 (HDRP(ΔNLS)). In a preferred embodiment, the variant thathybridizes under high stringency hybridizations encodes a polypeptidethat has a biological activity of an HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide (e.g., histone deacetylaseactivity or transcription repression activity).

Such nucleic acid molecules can be detected and/or isolated by specifichybridization (e.g., under high stringency conditions). “Specifichybridization,” as used herein, refers to the ability of a first nucleicacid to hybridize to a second nucleic acid in a manner such that thefirst nucleic acid does not hybridize to any nucleic acid other than tothe second nucleic acid (e.g., when the first nucleic acid has a highersimilarity to the second nucleic acid than to any other nucleic acid ina sample wherein the hybridization is to be performed). “Stringencyconditions” for hybridization is a term of art that refers to theincubation and wash conditions, e.g., conditions of temperature andbuffer concentration, that permit hybridization of a particular nucleicacid to a second nucleic acid; the first nucleic acid may be perfectly(i.e., 100%) complementary to the second, or the first and second mayshare some degree of complementarity that is less than perfect (e.g.,70%, 75%, 85%, 95%). For example, certain high stringency conditions canbe used that distinguish perfectly complementary nucleic acids fromthose of less complementarity. “High stringency conditions,” “moderatestringency conditions,” and “low stringency conditions” for nucleic acidhybridizations are explained on pages 2.10.1-2.10.16 and pages6.3.1-6.3.6 in Current Protocols in Molecular Biology (See Ausubel etal., supra, the entire teachings of which are incorporated by referenceherein). The exact conditions that determine the stringency ofhybridization depend not only on ionic strength (e.g., 0.2×SSC or0.1×SSC), temperature (e.g., room temperature, 42° C. or 68° C.), andthe concentration of destabilizing agents such as formamide ordenaturing agents such as SDS, but also on factors such as the length ofthe nucleic acid sequence, base composition, percent mismatch betweenhybridizing sequences, and the frequency of occurrence of subsets ofthat sequence within other non-identical sequences. Thus, equivalentconditions can be determined by varying one or more of these parameterswhile maintaining a similar degree of identity or similarity between thetwo nucleic acid molecules. Typically, conditions are used such thatsequences at least about 60%, at least about 70%, at least about 80%, atleast about 90% or at least about 95% or more identical to each otherremain hybridized to one another. By varying hybridization conditionsfrom a level of stringency at which no hybridization occurs to a levelat which hybridization is first observed, conditions that will allow agiven sequence to hybridize (e.g., selectively) with the most similarsequences in the sample can be determined.

Exemplary conditions are described in Krause and Aaronson, Methods inEnzymology, 200: 546-556 (1991). Also, in, Ausubel, et al., supra, whichdescribes the determination of washing conditions for moderate or lowstringency conditions. Washing is the step in which conditions areusually set so as to determine a minimum level of complementarity of thehybrids. Generally, starting from the lowest temperature at which onlyhomologous hybridization occurs, each ° C. by which the final washtemperature is reduced (holding SSC concentration constant) allows anincrease by 1% in the maximum extent of mismatching among the sequencesthat hybridize. Generally, doubling the concentration of SSC results inan increase in Tm of 17° C. Using these guidelines, the washingtemperature can be determined empirically for high, moderate, or lowstringency, depending on the level of mismatch sought.

For example, a low stringency wash can comprise washing in a solutioncontaining 0.2×SSC/0.1% SDS for 10 minutes at room temperature; amoderate stringency wash can comprise washing in a prewarmed solution(42° C.) solution containing 0.2×SSC/0.1% SDS for 15 minutes at 42° C.;and a high stringency wash can comprise washing in prewarmed (68° C.)solution containing 0.1×SSC/0.1% SDS for 15 minutes at 68° C.Furthermore, washes can be performed repeatedly or sequentially toobtain a desired result as known in the art. Equivalent conditions canbe determined by varying one or more of the parameters given as anexample, as known in the art, while maintaining a similar degree ofidentity or similarity between the target nucleic acid molecule and theprimer or probe used.

To determine the percent homology or identity of two nucleic acidsequences, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of one polypeptide ornucleic acid molecule for optimal alignment with the other polypeptideor nucleic acid molecule). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared, as described above.

The present invention also provides isolated HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), and HDRP(ΔNLS) nucleic acid molecules that contain afragment or portion that hybridizes under highly stringent conditions toa nucleotide sequence comprising a nucleotide sequence selected from SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, andthe complement of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQID NO: 7, or SEQ ID NO: 9 and also provides isolated nucleic acidmolecules that contain a fragment or portion that hybridizes underhighly stringent conditions to a nucleotide sequence encoding an aminoacid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, and SEQ ID NO: 10. The nucleic acid fragments of theinvention are at least about 15, preferably, at least about 18, 20, 23,or 25 nucleotides, and can be 30, 40, 50, 100, 200 or more nucleotidesin length. Longer fragments, for example, 30 or more nucleotides inlength, that encode antigenic polypeptides described herein areparticularly useful, such as for the generation of antibodies asdescribed above.

In a related aspect, the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), andHDRP(ΔNLS) nucleic acid fragments of the invention are used as probes orprimers in assays such as those described herein. “Probes” or “primers”are oligonucleotides that hybridize in a base-specific manner to acomplementary strand of nucleic acid molecules. Such probes and primersinclude polypeptide nucleic acids, as described in Nielsen et al.,Science, 254, 1497-1500 (1991). As also used herein, the term “primer”in particular refers to a single-stranded oligonucleotide that acts as apoint of initiation of template-directed DNA synthesis using well-knownmethods (e.g., PCR, LCR) including, but not limited to those describedherein.

Typically, a probe or primer comprises a region of nucleotide sequencethat hybridizes to at least about 15, typically about 20-25, and moretypically about 40, 50 or 75, consecutive nucleotides of a nucleic acidmolecule comprising a contiguous nucleotide sequence selected from: SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, thecomplement of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, and a sequence encoding an amino acid sequence ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO:10.

In preferred embodiments, a probe or primer comprises 100 or fewernucleotides, preferably, from 6 to 50 nucleotides, and more preferably,from 12 to 30 nucleotides. In other embodiments, the probe or primer isat least 70% identical to the contiguous nucleotide sequence or to thecomplement of the contiguous nucleotide sequence, preferably, at least80% identical, more preferably, at least 90% identical, even morepreferably, at least 95% identical, or even capable of selectivelyhybridizing to the contiguous nucleotide sequence or to the complementof the contiguous nucleotide sequence. Often, the probe or primerfurther comprises a label, e.g., radioisotope, fluorescent compound,enzyme, or enzyme co-factor.

The nucleic acid molecules of the invention such as those describedabove can be identified and isolated using standard molecular biologytechniques and the sequence information provided in SEQ ID NO: 1, SEQ IDNO; 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and/or SEQ ID NO: 10. For example,nucleic acid molecules can be amplified and isolated by the polymerasechain reaction using synthetic oligonucleotide primers designed based onone or more of the nucleic acid sequences provided above and/or thecomplement of those sequences. Or such nucleic acid molecules may bedesigned based on nucleotide sequences encoding one or more of the aminoacid sequences provided in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, or SEQ ID NO: 10. See generally PCR Technology: Principles andApplications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY,NY, (1992); PCR Protocols: A Guide to Methods and Applications (Eds.Innis et al., Academic Press, San Diego, Calif., (1990); Mattila et al.,Nucleic Acids Res., 19: 4967 (1991); Eckert et al., PCR Methods andApplications, 1:17 (1991); PCR (eds. McPherson et al., IRL Press,Oxford)); and U.S. Pat. No. 4,683,202. The nucleic acid molecules can beamplified using cDNA, mRNA, or genomic DNA as a template, cloned into anappropriate vector and characterized by DNA sequence analysis.

Other suitable amplification methods include the ligase chain reaction(LCR) (See Wu and Wallace, Genomics, 4:560 (1989), Landegren et al.,Science, 241:1077 (1988)), transcription amplification (Kwoh et al.,Proc. Natl. Acad. Sci. USA, 86:1173 (1989)), and self-sustained sequencereplication (See Guatelli et al., Proc. Nat. Acad. Sci. USA, 87:1874(1990)) and nucleic acid based sequence amplification (NASBA). Thelatter two amplification methods involve isothermal reactions based onisothermal transcription, that produce both single stranded RNA (ssRNA)and double stranded DNA (dsDNA) as the amplification products in a ratioof about 30 or 100 to 1, respectively.

The amplified DNA can be radiolabeled and used as a probe for screeninga cDNA library derived from human cells, mRNA in zap express, ZIPLOX, orother suitable vector. Corresponding clones can be isolated, DNA can beobtained following in vivo excision, and the cloned insert can besequenced in either or both orientations by art-recognized methods toidentify the correct reading frame encoding a polypeptide of theappropriate molecular weight. For example, the direct analysis of thenucleotide sequence of nucleic acid molecules of the present inventioncan be accomplished using well-known methods that are commerciallyavailable. See, for example, Sambrook et al., Molecular Cloning, ALaboratory Manual (2nd Ed., CSHP, New York (1989)); Zyskind et al.,Recombinant DNA Laboratory Manual, (Acad. Press, (1988)). Using these orsimilar methods, the polypeptide and the DNA encoding the polypeptidecan be isolated, sequenced, and further characterized.

Antisense nucleic acid molecules of the invention can be designed usingthe nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9 and/or the complement of any of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and/or a portionof those sequences, and/or the complement of those portion or sequences,and/or a sequence encoding the amino acid sequence of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or encoding aportion of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, orSEQ ID NO: 10. Such antisense nucleic acid molecules can be constructedusing chemical synthesis and enzymatic ligation reactions usingprocedures known in the art. For example, an antisense nucleic acidmolecule (e.g., an antisense oligonucleotide) can be chemicallysynthesized using naturally occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleic acids, e.g., phosphorothioatederivatives and acridine substituted nucleotides can be used.Alternatively, the antisense nucleic acid molecule can be producedbiologically using an expression vector into which a nucleic acidmolecule has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid molecule will be of anantisense orientation to a target nucleic acid of interest).

In general, the isolated HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), andHDRP(ΔNLS) nucleic acid sequences of the invention can be used asmolecular weight markers on Southern blots, and as chromosome markersthat are labeled to map related gene positions. The nucleic acidsequences can also be used to compare with endogenous DNA sequences inpatients to identify genetic disorders (e.g., a predisposition for orsusceptibility to a cell proliferation disease, an apoptotic disease, ora cell differentiation disease), and as probes, such as to hybridize anddiscover related DNA sequences or to subtract out known sequences from asample. The nucleic acid molecules of the present invention can also beused as therapeutic agents.

By a “cell proliferation disease” is meant a disease that is caused byor results in undesirably high levels of cell division, undesirably lowlevels of apoptosis, or both. For example, cancers such as lymphoma,leukemia, melanoma, ovarian cancer, breast cancer, pancreatic cancer,prostate cancer, colon cancer, and lung cancer are all examples of cellproliferation diseases. Myeloproliferative disorders, includingpolycythemia vera, essential thrombocythemia, agnogenic myeloidmetaplasia, and chronic myelogenous leukemia are also cell proliferationdiseases.

By a “cell differentiation disease” is meant a disease that is caused byor results in undesirably low levels of cell differentiation, or byundesirably high levels of cell differentiation. For example, cancerssuch as lymphoma, leukemia, melanoma, ovarian cancer, breast cancer,pancreatic cancer, prostate cancer, colon cancer, and lung cancer areall examples of cell differentiation diseases. Myeloproliferativedisorders, including polycythemia vera, essential thrombocythemia,agnogenic myeloid metaplasia, and chronic myelogenous leukemia are alsocell differentiation diseases.

By an “apoptotic disease” is meant a condition in which the apoptoticresponse is abnormal. This may pertain to a cell or a population ofcells that does not undergo cell death under appropriate conditions. Forexample, normally a cell will die upon exposure to apoptotic-triggeringagents, such as chemotherapeutic agents, or ionizing radiation. When,however, a subject has an apoptotic disease, for example, cancer, thecell or a population of cells may not undergo cell death in response tocontact with apoptotic-triggering agents. In addition, a subject mayhave an apoptotic disease when the occurrence of cell death is too low,for example, when the number of proliferating cells exceeds the numberof cells undergoing cell death, as occurs in cancer when such cells donot properly differentiate.

An apoptotic disease may also be a condition characterized by theoccurrence of undesirably high levels of apoptosis. For example, certainneurodegenerative diseases, including but not limited to Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, multiplesclerosis, restenosis, stroke, and ischemic brain injury are apoptoticdiseases in which neuronal cells undergo undesired cell death.

Other diseases for which the polypeptides and nucleic acid molecules ofthe present invention may be useful for diagnosing and/or treatinginclude, but are not limited to Huntington's disease.

The HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) nucleicacid molecules of the present invention can further be used to deriveprimers for genetic fingerprinting, to raise anti-polypeptide antibodiesusing DNA immunization techniques, and as an antigen to raise anti-DNAantibodies or elicit immune responses. Portions or fragments of thenucleotide sequences identified herein (and the corresponding completegene sequences) can be used in numerous ways as polynucleotide reagents.For example, these sequences can be used to: (i) map their respectivegenes on a chromosome; and, thus, locate gene regions associated withgenetic disease; (ii) identify an individual from a minute biologicalsample (tissue typing); and (iii) aid in forensic identification of abiological sample.

In addition, the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), andHDRP(ΔNLS) nucleotide sequences of the invention can be used to identifyand express recombinant polypeptides for analysis, characterization, ortherapeutic use, or as markers for tissues in which the correspondingpolypeptide is expressed, either constitutively, during tissuedifferentiation, or in diseased states. The nucleic acid sequences canadditionally be used as reagents in the screening and/or diagnosticassays described herein, and can also be included as components of kits(e.g., reagent kits) for use in the screening and/or diagnostic assaysdescribed herein.

Standard techniques, such as the polymerase chain reaction (PCR) and DNAhybridization, may be used to clone HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) homologs in other species, for example,mammalian homologs. HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) homologs may be readily identified using low-stringency DNAhybridization or low-stringency PCR with human HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) probes or primers. Degenerateprimers encoding human HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) polypeptides may be used to clone HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) homologs by RT-PCR.

Alternatively, additional HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) homologs can be identified by utilizing consensus sequenceinformation for HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)polypeptides to search for similar polypeptides in other species. Forexample, polypeptide databases for other species can be searched forproteins with the HDAC domains described herein. Candidate polypeptidescontaining such a motif can then be tested for their HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) biological activities, usingmethods described herein.

EXPRESSION OF THE NUCLEIC ACID MOLECULES OF THE INVENTION

Another aspect of the invention pertains to nucleic acid constructscontaining an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)nucleic acid molecule, for example, one selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, and the complement of any of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 (or portions thereof). Yetanother aspect of the invention pertains to HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), and HDRP(ΔNLS) nucleic acid constructs containing anucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. Theconstructs comprise a vector (e.g., an expression vector) into which asequence of the invention has been inserted in a sense or antisenseorientation.

As used herein, the term “vector” or “construct” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors, expressionvectors, are capable of directing the expression of genes to which theyare operably linked. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids. However,the invention is intended to include such other forms of expressionvectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses) that serveequivalent functions.

Preferred recombinant expression vectors of the invention comprise anucleic acid molecule of the invention in a form suitable for expressionof the nucleic acid molecule in a host cell. This means that therecombinant expression vectors include one or more regulatory sequences,selected on the basis of the host cells to be used for expression, whichis operably linked to the nucleic acid sequence to be expressed. Withina recombinant expression vector, “operably linked” is intended to meanthat the nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner that allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in ahost cell when the vector is introduced into the host cell). The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif., (1990). Regulatory sequences include those that directconstitutive expression of a nucleotide sequence in many types of hostcell and those that direct expression of the nucleotide sequence only incertain host cells (e.g., tissue-specific regulatory sequences).

It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed and the level of expression of polypeptidedesired. The expression vectors of the invention can be introduced intohost cells to thereby produce polypeptides, including fusionpolypeptides, encoded by nucleic acid molecules as described herein.

The recombinant expression vectors of the invention can be designed forexpression of a polypeptide of the invention in prokaryotic oreukaryotic cells, e.g., bacterial cells, such as E. coli, insect cells(using baculovirus expression vectors), yeast cells or mammalian cells.Suitable host cells are discussed further in Goeddel, supra.Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example, using T7 promoter regulatory sequencesand T7 polymerase.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, anucleic acid molecule of the invention can be expressed in bacterialcells (e.g., E. coli), insect cells, yeast, or mammalian cells (such asChinese hamster ovary cells (CHO) or COS cells, human 293T cells, HeLacells, NIH 3T3 cells, and mouse erythroleukemia (MEL) cells). Othersuitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing a foreign nucleicacid molecule (e.g., DNA) into a host cell, including calcium phosphateor calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, or electroporation. Suitable methods fortransforming or transfecting host cells can be found in Sambrook, et al.(supra), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those that confer resistance todrugs, such as G418, hygromycin, or methotrexate. Nucleic acid moleculesencoding a selectable marker can be introduced into a host cell on thesame vector as the nucleic acid molecule of the invention or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid molecule can be identified by drug selection(e.g., cells that have incorporated the selectable marker gene willsurvive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a polypeptide ofthe invention. Accordingly, the invention further provides methods forproducing a polypeptide using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of invention(into which a recombinant expression vector encoding a polypeptide ofthe invention has been introduced) in a suitable medium such that thepolypeptide is produced. In another embodiment, the method furthercomprises isolating the polypeptide from the medium or the host cell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into which anHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acidmolecule of the invention has been introduced. Such host cells can thenbe used to create non-human transgenic animals in which exogenousnucleotide sequences have been introduced into the genome or homologousrecombinant animals in which endogenous nucleotide sequences have beenaltered. Such animals are useful for studying the function and/oractivity of the nucleotide sequence and polypeptide encoded by thesequence and for identifying and/or evaluating modulators of theiractivity.

As used herein, a “transgenic animal” is a non-human animal, preferably,a mammal, more preferably, a rodent such as a rat or mouse, in which oneor more of the cells of the animal includes a transgene. Other examplesof transgenic animals include non-human primates, sheep, dogs, cows,goats, chickens, and amphibians. A transgene is exogenous DNA that isintegrated into the genome of a cell from which a transgenic animaldevelops and that remains in the genome of the mature animal, therebydirecting the expression, of an encoded gene product in one or more celltypes or tissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a non-human animal, preferably, a mammal, morepreferably, a mouse, in which an endogenous gene has been altered byhomologous recombination between the endogenous gene and an exogenousDNA molecule introduced into a cell of the animal, e.g., an embryoniccell of the animal, prior to development of the animal.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, U.S. Patent No. 4,873,191, and in Hogan,Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986)). Methods for constructing homologousrecombination vectors and homologous recombinant animals are describedfurther in Bradley, Current Opinion in Bio/Technology, 2: 823-829 (1991)and in PCT Publication Nos. WO 90/11354, WO 91/01140, WO 92/0968, and WO93/04169. Clones of the non-human transgenic animals described hereincan also be produced according to the methods described in Wilmut etal., Nature, 385: 810-813 (1997) and PCT Publication Nos. WO 97/07668and WO 97/07669.

ANTIBODIES OF THE INVENTION

Polyclonal and/or monoclonal antibodies that selectively bind one formof an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)polypeptide but not another form of the polypeptide are also provided.Antibodies are also provided that bind a portion of either the variantor reference HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)polypeptide that contains the polymorphic site or sites.

In another aspect, the invention provides antibodies to each of theHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) polypeptidesand polypeptide fragments of the invention, e.g., having an amino acidsequence encoded by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, or a portion thereof, or having an amino acid sequenceencoded by a nucleic acid molecule comprising all or a portion of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, (e.g.,SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO:10, or another variant, or portion thereof).

The term “purified antibody” as used herein refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site thatselectively binds an antigen. A molecule that selectively binds to apolypeptide of the invention is a molecule that binds to thatpolypeptide or a fragment thereof, but does not substantially bind othermolecules in a sample, e.g., a biological sample that naturally containsthe polypeptide. Preferably the antibody is at least 60%, by weight,free from proteins and naturally occurring organic molecules with whichit naturally associated. More preferably, the antibody preparation is atleast 75% or 90%, and most preferably, 99%, by weight, antibody.Examples of immunologically active portions of immunoglobulin moleculesinclude F(ab) and F(ab′)2 fragments that can be generated by treatingthe antibody with an enzyme such as pepsin.

The invention provides polyclonal and monoclonal antibodies thatselectively bind to an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) polypeptide of the invention. The term “monoclonal antibody”or “monoclonal antibody composition,” as used herein, refers to apopulation of antibody molecules that contain only one species of anantigen binding site capable of immunoreacting with a particular epitopeof a polypeptide of the invention. A monoclonal antibody compositionthus typically displays a single binding affinity for a particularpolypeptide of the invention with which it immunoreacts.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a desired immunogen, e.g., an HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide of the invention orfragment thereof. The antibody titer in the immunized subject can bemonitored over time by standard techniques, such as with an enzymelinked immunosorbent assay (ELISA) using immobilized polypeptide. Ifdesired, the antibody molecules directed against the polypeptide can beisolated from the mammal (e.g., from the blood) and further purified bywell-known techniques, such as protein A chromatography to obtain theIgG fraction.

At an appropriate time after immunization, e.g., when the antibodytiters are highest, antibody-producing cells can be obtained from thesubject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein, Nature, 256: 495-497 (1975), the human B cellhybridoma technique (Kozbor et al., Immunol. Today, 4: 72 (1983)), theEBV-hybridoma technique (Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96 (1985)) or trioma techniques. Thetechnology for producing hybridomas is well known (see generally CurrentProtocols in Immunology, Coligan et al., (eds.) John Wiley & Sons, Inc.,New York, N.Y., (1994)). Briefly, an immortal cell line (typically amyeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with an immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds a polypeptide ofthe invention.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating amonoclonal antibody to a polypeptide of the invention (see, e.g.,Current Protocols in Immunology, supra; Galfre et al., (1977) Nature,266:55052; R. H. Kenneth, in Monoclonal Antibodies: A New Dimension InBiological Analyses, Plenum Publishing Corp., New York, N.Y., (1980);and Lerner, Yale J. Biol. Med., 54: 387-402 (1981)). Moreover, theordinarily skilled worker will appreciate that there are many variationsof such methods that also would be useful.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody to an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) polypeptide of the invention can be identified and isolatedby screening a recombinant combinatorial immunoglobulin library (e.g.,an antibody phage display library) with the polypeptide to therebyisolate immunoglobulin library members that bind the polypeptide. Kitsfor generating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCTPublication No. WO 92/20791; PCT Publication No. WO 92/15679; PCTPublication No. WO 93/01288; PCT Publication No. WO 92/01047; PCTPublication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs etal., Bio/Technology, 9: 1370-1372 (1991); Hay et al., Hum. Antibod.Hybridomas, 3: 81-85 (1992); Huse et al., Science, 246: 1275-1281(1989); and Griffiths et al., EMBO J., 12: 725-734 (1993).

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the invention. Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart.

In general, antibodies of the invention (e.g., a monoclonal antibody)can be used to isolate an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) polypeptide of the invention by standard techniques, such asaffinity chromatography or immunoprecipitation. A polypeptide-specificantibody can facilitate the purification of natural polypeptide fromcells and of recombinantly produced polypeptide expressed in host cells.Moreover, an antibody specific for an HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide of the invention can be used todetect the polypeptide (e.g., in a cellular lysate, cell supernatant, ortissue sample) in order to evaluate the abundance and pattern ofexpression of the polypeptide.

The antibodies of the present invention can also be used diagnosticallyto monitor protein levels in tissue as part of a clinical testingprocedure, e.g., to, for example, determine the efficacy of a giventreatment regimen. Detection can be facilitated by coupling the antibodyto a detectable substance. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, and acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin;an example of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequofin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S,and ³H.

DIAGNOSTIC AND SCREENING ASSAYS OF THE INVENTION

The present invention also pertains to diagnostic assays for assessingHDAC9 HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) gene expression,or for assessing activity of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS),or HDRP(ΔNLS) polypeptides of the invention. In one embodiment, theassays are used in the context of a biological sample (e.g., blood,serum, cells, tissue) to thereby determine whether an individual isafflicted with a cell proliferation disease, an apoptotic disease, or acell differentiation disease, or is at risk for (has a predispositionfor or a susceptibility to) developing a cell proliferation disease, anapoptotic disease, or a cell differentiation disease. The invention alsoprovides for prognostic (or predictive) assays for determining whetheran individual is susceptible to developing a cell proliferation disease,an apoptotic disease, or a cell differentiation disease. For example,mutations in the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)nucleic acid molecule can be assayed in a biological sample. Such assayscan be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset of symptomsassociated with a cell proliferation disease, an apoptotic disease, or acell differentiation disease.

Another aspect of the invention pertains to assays for monitoring theinfluence of agents, or candidate compounds (e.g., drugs or otheragents) on the nucleic acid molecule expression or biological activityof polypeptides of the invention, as well as to assays for identifyingcandidate compounds that bind to an HDAC9, HDAC9a polypeptide, anHDAC9(ΔNLS) polypeptide, an HDAC9a(ΔNLS) polypeptide, or an HDRP(ΔNLS)polypeptide. These and other assays and agents are described in furtherdetail in the following sections.

DIAGNOSTIC ASSAYS

HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acidmolecules, probes, primers, polypeptides, and antibodies to an HDAC9, anHDAC9a protein, an HDAC9(ΔNLS) protein, an HDAC9a(ΔNLS) protein, or anHDRP(ΔNLS) protein can be used in methods of diagnosis of asusceptibility to, or likelihood of having a cell proliferation disease,an apoptotic disease, or a cell differentiation disease, as well as inkits useful for diagnosis of a susceptibility to a cell proliferationdisease, an apoptotic disease, or a cell differentiation disease.

In one embodiment of the invention, diagnosis of a decreasedsusceptibility to a cell proliferation disease, an apoptotic disease, ora cell differentiation disease is made by detecting a polymorphism inHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). Thepolymorphism can be a mutation in HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS), such as the insertion or deletion of asingle nucleotide, or of more than one nucleotide, resulting in a frameshift mutation; the change of at least one nucleotide, resulting in achange in the encoded amino acid; the change of at least one nucleotide,resulting in the generation of a premature stop codon; the deletion ofseveral nucleotides, resulting in a deletion of one or more amino acidsencoded by the nucleotides; the insertion of one or several nucleotides,such as by unequal recombination or gene conversion, resulting in aninterruption of the coding sequence of the gene; duplication of all or apart of the gene; transposition of all or a part of the gene; orrearrangement of all or a part of the gene, or a change in theexpression pattern of the various HDAC9 isoforms. More than one suchmutation may be present in a single nucleic acid molecule.

Such sequence changes cause a mutation in the polypeptide encoded byHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). For example, ifthe mutation is a frame shift mutation, the frame shift can result in achange in the encoded amino acids, and/or can result in the generationof a premature stop codon, causing generation of a truncatedpolypeptide. Alternatively, a polymorphism associated with a decreasedsusceptibility to a cell proliferation disease, an apoptotic disease, ora cell differentiation disease can be a synonymous mutation in one ormore nucleotides (i.e., a mutation that does not result in a change inthe HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)polypeptide). Such a polymorphism may alter sites, affect the stabilityor transport of mRNA, or otherwise affect the transcription ortranslation of the nucleic acid molecule. HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) that has any of the mutations describedabove is referred to herein as a “mutant nucleic acid molecule.”

In a first method of diagnosing a decreased susceptibility to a cellproliferation disease, an apoptotic disease, or a cell differentiationdisease, hybridization methods, such as Southern analysis, Northernanalysis, or in situ hybridizations, can be used (see Ausubel, et al.,supra). For example, a biological sample from a test subject (a “testsample”) of genomic DNA, RNA, or cDNA, is obtained from an individualsuspected of having, being susceptible to or predisposed for, orcarrying a defect for, a cell proliferation disease, an apoptoticdisease, or a cell differentiation disease (the “test individual”). Theindividual can be an adult, child, or fetus. The test sample can be fromany source that contains genomic DNA, such as a blood sample, sample ofamniotic fluid, sample of cerebrospinal fluid, or tissue sample fromskin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinaltract, or other organs. A test sample of DNA from fetal cells or tissuecan be obtained by appropriate methods, such as by amniocentesis orchorionic villus sampling. The DNA, RNA, or cDNA sample is then examinedto determine whether a polymorphism in HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) is present, and/or to determine whichvariant(s) encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) is present. The presence of the polymorphism or variant(s)can be indicated by hybridization of the gene in the genomic DNA, RNA,or cDNA to a nucleic acid probe. A “nucleic acid probe,” as used herein,can be a DNA probe or an RNA probe; the nucleic acid probe can containat least one polymorphism in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS),or HDRP(ΔNLS) or contains a nucleic acid encoding a particular variantof HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). The probecan be any of the nucleic acid molecules described above (e.g., theentire nucleic acid molecule, a fragment, a vector comprising the gene,a probe, or primer, etc.).

To diagnose a decreased susceptibility to a cell proliferation disease,an apoptotic disease, or a cell differentiation disease, a hybridizationsample is formed by contacting the test sample containing HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), with at least one nucleic acidprobe. A preferred probe for detecting mRNA or genomic DNA is a labelednucleic acid probe capable of hybridizing to HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) mRNA or genomic DNA sequences describedherein. The nucleic acid probe can be, for example, a full-lengthnucleic acid molecule, or a portion thereof, such as an oligonucleotideof at least 15, 30, 50, 100, 250, or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions toappropriate mRNA or genomic DNA. For example, the nucleic acid probe canbe all or a portion of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, or the complement of SEQ ID NO: 1 or SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9; or can be a nucleic acidmolecule encoding all or a portion of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. Other suitable probes for use inthe diagnostic assays of the invention are described above (see. e.g.,probes and primers discussed under the heading, “Nucleic Acids of theInvention”).

The hybridization sample is maintained under conditions that aresufficient to allow specific hybridization of the nucleic acid probe toHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). “Specifichybridization,” as used herein, indicates exact hybridization (e.g.,with no mismatches). Specific hybridization can be performed under highstringency conditions or moderate stringency conditions, for example, asdescribed above. In a particularly preferred embodiment, thehybridization conditions for specific hybridization are high stringency.

Specific hybridization, if present, is then detected using standardmethods. If specific hybridization occurs between the nucleic acid probeand HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) in the testsample, then HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) hasthe polymorphism, or is the variant, that is present in the nucleic acidprobe. More than one nucleic acid probe can also be used concurrently inthis method. Specific hybridization of any one of the nucleic acidprobes is indicative of a polymorphism in HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS), or of the presence of a particular variantencoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), andis therefore diagnostic for a decreased susceptibility to a cellproliferation disease, an apoptotic disease, or a cell differentiationdisease.

In Northern analysis (see Current Protocols in Molecular Biology,Ausubel, et al., supra), the hybridization methods described above areused to identify the presence of a polymorphism or of a particularvariant, associated with a decreased susceptibility to a cellproliferation disease, an apoptotic disease, or a cell differentiationdisease. For Northern analysis, a test sample of RNA is obtained fromthe individual by appropriate means. Specific hybridization of a nucleicacid probe, as described above, to RNA from the individual is indicativeof a polymorphism in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS), or of the presence of a particular variant encoded by HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), and is thereforediagnostic for a decreased susceptibility to a cell proliferationdisease, an apoptotic disease, or a cell differentiation disease.

For representative examples of use of nucleic acid probes, see, forexample, U.S. Pat. Nos. 5,288,611 and 4,851,330.

Alternatively, a peptide nucleic acid (PNA) probe can be used instead ofa nucleic acid probe in the hybridization methods described above. PNAis a DNA mimic having a peptide-like, inorganic backbone, such asN-(2-aminoethyl)glycine units, with an organic base (A, G, C, T, or U)attached to the glycine nitrogen via a methylene carbonyl linker (see,for example, Nielsen et al., Bioconjugate Chemistry, 5 (1994), AmericanChemical Society, p. 1 (1994)). The PNA probe can be designed tospecifically hybridize to a gene having a polymorphisrm associated witha susceptibility to a cell proliferation disease, an apoptotic disease,or a cell differentiation disease. Hybridization of the PNA probe toHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) is diagnosticfor a decreased susceptibility to a cell proliferation disease, anapoptotic disease, or a cell differentiation disease.

In another method of the invention, mutation analysis by restrictiondigestion can be used to detect a mutant nucleic acid molecule, ornucleic acid molecules containing a polymorphism(s), if the mutation orpolymorphism in the gene results in the creation or elimination of arestriction site. A test sample containing genomic DNA is obtained fromthe individual. Polymerase chain reaction (PCR) can be used to amplifyHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) (and, ifnecessary, the flanking sequences) in the test sample of genomic DNAfrom the test individual. RFLP analysis is conducted as described (seeCurrent Protocols in Molecular Biology, supra). The digestion pattern ofthe relevant DNA fragment indicates the presence or absence of themutation or polymorphism in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS), and therefore indicates the presence or absence of thisdecreased susceptibility to a cell proliferation disease, an apoptoticdisease, or a cell differentiation disease.

Sequence analysis can also be used to detect specific polymorphisms inHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). A test sampleof DNA or RNA is obtained from the test individual. PCR or otherappropriate methods can be used to amplify the nucleic acid molecule,and/or its flanking sequences, if desired. The sequence of HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), or HDRP(ΔNLS), or afragment of the any of those nucleic acid molecules, or an HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) cDNA, or a fragment ofany of those cDNAs, or an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) mRNA, or a fragment of any of those mRNAs, is determined,using standard methods. The sequence of the above gene, gene fragment,cDNA, cDNA fragment, mRNA, or mRNA fragment is compared with the knownnucleic acid sequence of the nucleic acid molecule, cDNA (e.g., SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or anucleic acid sequence encoding the protein of SEQ ID NO: 2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, or a fragment thereof) or mRNA,as appropriate. The presence of a polymorphism in HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) indicates that the individualhas a decreased susceptibility to a cell proliferation disease, anapoptotic disease, or a cell differentiation disease.

Allele-specific oligonucleotides can also be used to detect the presenceof a polymorphism in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS), through the use of dot-blot hybridization of amplifiedoligonucleotides with allele-specific oligonucleotide (ASO) probes (see,for example, Saiki et al., Nature (London) 324: 163-166 (1986)). An“allele-specific oligonucleotide” (also referred to herein as an“allele-specific oligonucleotide probe”) is an oligonucleotide ofapproximately 10-50 base pairs, preferably approximately 15-30 basepairs, that specifically hybridizes to HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS), and that contains a polymorphism associatedwith a decreased susceptibility to a cell proliferation disease, anapoptotic disease, or a cell differentiation disease. An allele-specificoligonucleotide probe that is specific for particular polymorphisms inHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) can be prepared,using standard methods (see Current Protocols in Molecular Biology,supra).

To identify polymorphisms in the gene that are associated with adecreased susceptibility to a cell proliferation disease, an apoptoticdisease, or a cell differentiation disease a test sample of DNA isobtained from the individual. PCR can be used to amplify all or afragment of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), andits flanking sequences. The DNA containing the amplified HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) (or a fragment of any of thosegenes) is dot-blotted, using standard methods (see Current Protocols inMolecular Biology, supra), and the blot is contacted with theoligonucleotide probe. The presence of specific hybridization of theprobe to the amplified HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) is then detected. Specific hybridization of anallele-specific oligonucleotide probe to DNA from the individual isindicative of a polymorphism in HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS), and is therefore indicative of a decreasedsusceptibility to a cell proliferation disease, an apoptotic disease, ora cell differentiation disease.

In another embodiment, arrays of oligonucleotide probes that arecomplementary to target nucleic acid sequence segments from anindividual, can be used to identify polymorphisms in HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). For example, in oneembodiment, an oligonucleotide array can be used. Oligonucleotide arraystypically comprise a plurality of different oligonucleotide probes thatare coupled to a surface of a substrate in different known locations.These oligonucleotide arrays, also described as “GENECHIPS™,” have beengenerally described in the art, for example, U.S. Pat. No. 5,143,854 andPCT patent publication Nos. WO 90/15070 and 92/10092. These arrays cangenerally be produced using mechanical synthesis methods or lightdirected synthesis methods that incorporate a combination ofphotolithographic methods and solid phase oligonucleotide synthesismethods. See Fodor et al., Science, 251: 767-777 (1991), Pirrung et al.,U.S. Pat. No. 5,143,854; PCT Publication No. WO 90/15070; Fodor et al.,PCT Publication No. WO 92/10092, and U.S. Pat. No. 5,424,186, the entireteachings of each of which are incorporated by reference herein.Techniques for the synthesis of these arrays using mechanical synthesismethods are described in, e.g., U.S. Pat. No. 5,384,261, the entireteachings of which are incorporated by reference herein.

Once an oligonucleotide array is prepared, a nucleic acid of interest ishybridized to the array and scanned for polymorphisms. Hybridization andscanning are generally carried out by methods described herein and alsoin, e.g., Published PCT Application Nos. WO 92/10092 and WO 95/11995,and U.S. Pat. No. 5,424,186, the entire teachings of which areincorporated by reference herein. In brief, a target nucleic acidsequence that includes one or more previously identified polymorphicmarkers is amplified by well known amplification techniques, e.g., PCR.Typically, this involves the use of primer sequences that arecomplementary to the two strands of the target sequence both upstreamand downstream from the polymorphism. Asymmetric PCR techniques may alsobe used. Amplified target, generally incorporating a label, is thenhybridized with the array under appropriate conditions. Upon completionof hybridization and washing of the array, the array is scanned todetermine the position on the array to which the target sequencehybridizes. The hybridization data obtained from the scan is typicallyin the form of fluorescence intensities as a function of location on thearray.

Although primarily described in terms of a single detection block, e.g.,for detection of a single polymorphism, arrays can include multipledetection blocks, and thus be capable of analyzing multiple, specificpolymorphisms. In alternate arrangements, it will generally beunderstood that detection blocks may be grouped within a single array orin multiple, separate arrays so that varying, optimal conditions may beused during the hybridization of the target to the array. For example,it may often be desirable to provide for the detection of thosepolymorphisms that fall within G-C rich stretches of a genomic sequence,separately from those falling in A-T rich segments. This allows for theseparate optimization of hybridization conditions for each situation.

Additional descriptions of the use of oligonucleotide arrays fordetection of polymorphisms can be found, for example, in U.S. Pat. Nos.5,858,659 and 5,837,832, the entire teachings of which are incorporatedby reference herein.

Other methods of nucleic acid analysis can be used to detectpolymorphisms in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)or variants encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS). Representative methods include direct manual sequencing(Church and Gilbert Proc. Natl. Acad. Sci. USA 81: 1991-1995, (1988);Sanger et al., Proc. Natl. Acad. Sci. 74: 5463-5467 (1977); Beavis etal., U.S. Pat. No. 5,288,644); automated fluorescent sequencing;single-stranded conformation polymorphism assays (SSCP); clampeddenaturing gel electrophoresis (CDGE); denaturing gradient gelelectrophoresis (DGGE) (Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-236 (1991)), mobility shift analysis (Orita et al., Proc. Natl.Acad. Sci. USA 86: 2766-2770 (1989)), restriction enzyme analysis(Flavell et al., Cell 15: 25 (1978); Geever, et al., Proc. Natl. Acad.Sci. USA 78: 5081 (1981)); heteroduplex analysis; chemical mismatchcleavage (CMC) (Cotton et al., Proc. Natl. Acad. Sci. USA 85: 4397-4401(1985)); RNase protection assays (Myers et al., Science 230: 1242(1985)); use of polypeptides that recognize nucleotide mismatches, suchas E. coli mutS protein; and allele-specific PCR.

In another embodiment of the invention, diagnosis of a susceptibility toa cell proliferation disease, an apoptotic disease, or a celldifferentiation disease can also be made by examining the level of anHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid,for example, using in situ hybridization techniques known to one skilledin the art, or by examining the level of expression, activity, and/orcomposition of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) polypeptide, by a variety of methods, including enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations,immunohistochemistry, and immunofluorescence. A test sample from anindividual is assessed for the presence of an alteration in the level ofan HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acidor in the expression and/or an alteration in composition of thepolypeptide encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS), or for the presence of a particular variant encoded byHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). An alterationin expression of a polypeptide encoded by HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) can be, for example, an alteration in thequantitative polypeptide expression (i.e., the amount of polypeptideproduced); an alteration in the composition of a polypeptide encoded byHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), or analteration in the qualitative polypeptide expression (e.g., expressionof a mutant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)polypeptide or variant thereof). In a preferred embodiment, diagnosis ofa susceptibility to a cell proliferation disease, an apoptotic disease,or a cell differentiation disease is made by detecting a particularvariant encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS), or a particular pattern of variants. Preferably, increasedlevels of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) orincreased expression or activity of an HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, relative to a control sample,for example, a sample known not to be associated with a cellproliferation disease, an apoptotic disease, or a cell differentiationdisease, indicates an increased susceptibility or likelihood that theindividual has a cell proliferation disease, an apoptotic disease, or acell differentiation disease. Alternatively, decreased levels of HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) or decreased expressionor activity of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) polypeptide, relative to a control sample, for example, asample known not to be associated with a cell proliferation disease, anapoptotic disease, or a cell differentiation disease, indicates adecreased susceptibility or likelihood that the individual has a cellproliferation disease, an apoptotic disease, or a cell differentiationdisease.

Both quantitative and qualitative alterations can also be present. An“alteration” or “modulation” in the polypeptide expression, activity, orcomposition, as used herein, refers to an alteration in expression orcomposition in a test sample, as compared with the expression orcomposition of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)polypeptide in a control sample. A control sample is a sample thatcorresponds to the test sample (e.g., is from the same type of cells),and is from an individual who is not affected by a cell proliferationdisease, an apoptotic disease, or a cell differentiation disease. Analteration in the expression or composition of the polypeptide in thetest sample, as compared with the control sample, is indicative of adecreased susceptibility to a cell proliferation disease, an apoptoticdisease, or a cell differentiation disease. Similarly, the presence ofone or more different variants in the test sample, or the presence ofsignificantly different amounts of different variants in the testsample, as compared with the control sample, is indicative of adecreased susceptibility to a cell proliferation disease, an apoptoticdisease, or a cell differentiation disease.

It is understood that alterations or modulations in polypeptideexpression or function can occur in varying degrees. For example, analteration or modulation in expression can be an increase, for example,by at least 1.5-fold to 2-fold, at least 3-fold, or, at least 5-fold,relative to the control. Alternatively, the alteration or modulation inpolypeptide expression can be a decrease, for example, by at least 10%,at least 40%, 50%, or 75%, or by at least 90%, relative to the control.

Various means of examining expression or composition of the HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide can beused, including spectroscopy, colorimetry, electrophoresis, isoelectricfocusing, and immunoassays (e.g., David et al., U.S. Pat. No. 4,376,110)such as immunoblotting (see also Ausubel et al., supra; particularlychapter 10). For example, in one embodiment, an antibody capable ofbinding to the polypeptide (e.g., as described above), preferably anantibody with a detectable label, can be used. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled,”with regard to the antibody, is intended to encompass direct labeling ofthe antibody by coupling (i.e., physically linking) a detectablesubstance to the antibody, as well as indirect labeling of the antibodyby reacting it with another reagent that is directly labeled. An exampleof indirect labeling is detection of a primary antibody using afluorescently labeled secondary antibody.

Western blotting analysis, using an antibody as described above thatspecifically binds to a mutant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS),or HDRP(ΔNLS) polypeptide, or an antibody that specifically binds to anon-mutant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)polypeptide, or an antibody that specifically binds to a particularvariant encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS), can be used to identify the presence in a test sample of aparticular variant of a polypeptide encoded by a polymorphic or mutantHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), or the absencein a test sample of a particular variant or of a polypeptide encoded bya non-polymorphic or non-mutant gene. The presence of a polypeptideencoded by a polymorphic or mutant gene, or the absence of a polypeptideencoded by a non-polymorphic or non-mutant gene, is diagnostic for adecreased susceptibility to a cell proliferation disease, an apoptoticdisease, or a cell differentiation disease, as is the presence (orabsence) of particular variants encoded by the HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule.

In one embodiment of this method, the level or amount of HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide in a test sample iscompared with the level or amount of the HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide in a control sample. A level oramount of the polypeptide in the test sample that is higher or lowerthan the level or amount of the polypeptide in the control sample, suchthat the difference is statistically significant, is indicative of analteration in the expression of the HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, and is diagnostic for adecreased susceptibility to a cell proliferation disease, an apoptoticdisease, or a cell differentiation disease.

Alternatively, the composition of the HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide in a test sample is comparedwith the composition of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) polypeptide in a control sample. A difference in thecomposition of the polypeptide in the test sample, as compared with thecomposition of the polypeptide in the control sample (e.g., the presenceof different variants), is diagnostic for a decreased susceptibility toa cell proliferation disease, an apoptotic disease, or a celldifferentiation disease. In another embodiment, both the level or amountand the composition of the polypeptide can be assessed in the testsample and in the control sample. A difference in the amount or level ofthe polypeptide in the test sample, compared to the control sample; adifference in composition in the test sample, compared to the controlsample; or both a difference in the amount or level, and a difference inthe composition, is indicative of a decreased susceptibility to a cellproliferation disease, an apoptotic disease, or a cell differentiationdisease.

Kits (e.g., reagent kits) useful in the methods of diagnosis comprisecomponents useful in any of the methods described herein, including, forexample, hybridization probes or primers as described herein (e.g.,labeled probes or primers), reagents for detection of labeled molecules,restriction enzymes (e.g., for RFLP analysis), allele-specificoligonucleotides, antibodies that bind to a mutant or to non-mutant(native) HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)polypeptide, means for amplification of nucleic acids comprising HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), or means for analyzingthe nucleic acid sequence of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS),or HDRP(ΔNLS), or for analyzing the amino acid sequence of an HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, etc.

SCREENING ASSAYS AND AGENTS IDENTIHED THEREBY

The invention provides methods (also referred to herein as “screeningassays”) for identifying the presence of a nucleotide that hybridizes toa nucleic acid of the invention, as well as for identifying the presenceof a polypeptide encoded by a nucleic acid of the invention. In oneembodiment, the presence (or absence) of a nucleic acid molecule ofinterest (e.g., a nucleic acid that has significant homology with anucleic acid of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS))in a sample can be assessed by contacting the sample with a nucleic acidcomprising a nucleic acid of the invention (e.g., a nucleic acid havingthe sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,or SEQ ID NO: 9, which may optionally comprise at least onepolymorphism, or the complement thereof, or a nucleic acid encoding anamino acid having the sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO: 8, or SEQ ID NO: 10, or a fragment or variant of suchnucleic acids), under stringent conditions as described above, and thenassessing the sample for the presence (or absence) of hybridization. Ina preferred embodiment, high stringency conditions are conditionsappropriate for selective hybridization. In another embodiment, a samplecontaining the nucleic acid molecule of interest is contacted with anucleic acid containing a contiguous nucleotide sequence (e.g., a primeror a probe as described above) that is at least partially complementaryto a part of the nucleic acid molecule of interest (e.g., an HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid), and thecontacted sample is assessed for the presence or absence ofhybridization. In a preferred embodiment, the nucleic acid containing acontiguous nucleotide sequence is completely complementary to a part ofthe nucleic acid molecule of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS),or HDRP(ΔNLS).

In any of the above embodiments, all or a portion of the nucleic acid ofinterest can be subjected to amplification prior to performing thehybridization.

In another embodiment, the presence (or absence) of an HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, such as apolypeptide of the invention or a fragment or variant thereof, in asample can be assessed by contacting the sample with an antibody thatspecifically binds to the polypeptide of HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) (e.g., an antibody such as those describedabove), and then assessing the sample for the presence (or absence) ofbinding of the antibody to the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS),or HDRP(ΔNLS) polypeptide.

In another embodiment, the invention provides methods for identifyingagents or compounds (e.g., fusion proteins, polypeptides,peptidomimetics, prodrugs, receptors, binding agents, antibodies, smallmolecules or other drugs, or ribozymes) that alter or modulate (e.g.,increase or decrease) the activity of the polypeptides described herein,or that otherwise interact with the polypeptides herein. For example,such compounds can be compounds or agents that bind to polypeptidesdescribed herein (e.g., HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) substrates or agents); that have a stimulatory or inhibitoryeffect on, for example, activity of polypeptides of the invention; orthat change (e.g., enhance or inhibit) the ability of the polypeptidesof the invention to interact with HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) binding agents; or that alterpost-translational processing of the HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide (e.g., agents that alterproteolytic processing to direct the polypeptide from where it isnormally synthesized to another location in the cell, such as the cellsurface; or agents that alter proteolytic processing such that morepolypeptide is released from the cell, etc.). In one example, thebinding agent is a cell proliferation disease binding agent, anapoptotic disease binding agent, or a cell differentiation diseasebinding agent. As used herein, by a “cell proliferation disease bindingagent,” an “apoptotic disease binding agent,” or a “cell differentiationdisease binding agent” is meant an agent as described herein that bindsto a polypeptide of the present invention and modulates a cellproliferation disease, an apoptotic disease, or a cell differentiationdisease. The modulation can be an increase or a decrease in the severityor progression of the disease. In addition, a cell proliferation diseasebinding agent, an apoptotic disease binding agent, or a celldifferentiation disease binding agent includes an agent that binds to apolypeptide that is upstream (earlier) or downstream (later) of the cellsignaling events mediated by a polypeptide of the present invention, andthereby modulates the overall activity of the signaling pathway; inturn, the disease state is modulated.

The candidate compound can cause an increase in the activity of thepolypeptide. For example, the activity of the polypeptide can beincreased by at least 1.5-fold to 2-fold, at least 3-fold, or, at least5-fold, relative to the control. Alternatively, the polypeptide activitycan be a decrease, for example, by at least 10%, at least 20%, 40%, 50%,or 75%, or by at least 90%, relative to the control.

In one embodiment, the invention provides assays for screening candidatecompounds or test agents to identify compounds that bind to or modulatethe activity of polypeptides described herein (or biologically activeportion(s) thereof), as well as agents identifiable by the assays. Asused herein, a “candidate compound” or “test agent” is a chemicalmolecule, be it naturally-occurring or artificially-derived, andincludes, for example, peptides, proteins, synthesized molecules, forexample, synthetic organic molecules, naturally-occurring molecule, forexample, naturally occurring organic molecules, nucleic acid molecules,and components thereof.

In general, candidate compounds for uses in the present invention may beidentified from large libraries of natural products or synthetic (orsemi-synthetic) extracts or chemical libraries according to methodsknown in the art. Those skilled in the field of drug discovery anddevelopment will understand that the precise source of test extracts orcompounds is not critical to the screening procedure(s) of theinvention. Accordingly, virtually any number of chemical extracts orcompounds can be screened using the exemplary methods described herein.Examples of such extracts or compounds include, but are not limited to,plant-, fungal-, prokaryotic- or animal-based extracts, fermentationbroths, and synthetic compounds, as well as modification of existingcompounds. Numerous methods are also available for generating random ordirected synthesis (e.g., semi-synthesis or total synthesis) of anynumber of chemical compounds, including, but not limited to,saccharide-, lipid-, peptide-, and nucleic acid-based compounds.Synthetic compound libraries are commercially available, e.g., fromBrandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee,Wis.). Alternatively, libraries of natural compounds in the form ofbacterial, fungal, plant, and animal extracts are commercially availablefrom a number of sources, including Biotics (Sussex, UK), Xenova(Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.),and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural andsynthetically produced libraries are generated, if desired, according tomethods known in the art, e.g., by standard extraction and fractionationmethods. For example, candidate compounds can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries, synthetic library methodsrequiring deconvolution; the “one-bead one-compound” library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to polypeptide libraries, whilethe other four approaches are applicable to polypeptide, non-peptideoligomer or small molecule libraries of compounds (Lam, Anticancer DrugDes., 12: 145 (1997)). Furthermore, if desired, any library or compoundis readily modified using standard chemical, physical, or biochemicalmethods.

In addition, those skilled in the art of drug discovery and developmentreadily understand that methods for dereplication (e.g., taxonomicdereplication, biological dereplication, and chemical dereplication, orany combination thereof) or the elimination of replicates or repeats ofmaterials already known for their activities should be employed wheneverpossible.

When a crude extract is found to modulate (i.e., stimulate or inhibit)the expression and/or activity of the nucleic acids and or polypeptidesof the present invention, further fractionation of the positive leadextract is necessary to isolate chemical constituents responsible forthe observed effect. Thus, the goal of the extraction, fractionation,and purification process is the careful characterization andidentification of a chemical entity within the crude extract having anactivity that stimulates or inhibits nucleic acid expression,polypeptide expression, or polypeptide biological activity. The sameassays described herein for the detection of activities in mixtures ofcompounds can be used to purify the active component and to testderivatives thereof. Methods of fractionation and purification of suchheterogenous extracts are known in the art. If desired, compounds shownto be useful agents for treatment are chemically modified according tomethods known in the art. Compounds identified as being of therapeuticvalue may be subsequently analyzed using animal models for diseases inwhich it is desirable to alter the activity or expression of the nucleicacids or polypeptides of the present invention.

In one embodiment, to identify candidate compounds that alter thebiological activity, for example, the enzymatic activity ortranscriptional repression activity of an HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, a cell, tissue, cell lysate,tissue lysate, or solution containing or expressing an HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide (e.g., SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SE ID NO: 8, SEQ ID NO: 10, or anothervariant encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS)), or a fragment or derivative thereof (as described above),can be contacted with a candidate compound to be tested under conditionssuitable for enzymatic reaction or transcriptional repression reaction,as described herein.

Alternatively, the polypeptide can be contacted directly with thecandidate compound to be tested. The level (amount) of HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) biological activity is assessed(e.g., the level (amount) of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS),or HDRP(ΔNLS) biological activity is measured, either directly orindirectly), and is compared with the level of biological activity in acontrol (i.e., the level of activity of the HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide or active fragment or derivativethereof in the absence of the candidate compound to be tested, or in thepresence of the candidate compound vehicle only). If the level of thebiological activity in the presence of the candidate compound differs,by an amount that is statistically significant, from the level of thebiological activity in the absence of the candidate compound, or in thepresence of the candidate compound vehicle only, then the candidatecompound is a compound that alters the biological activity of an HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide. Forexample, an increase in the level of HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) enzymatic or transcriptional repressionactivity relative to a control, indicates that the candidate compound isa compound that enhances (is an agonist of) HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) activity. Similarly, a decrease in theenzymatic level or transcriptional repression level of HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) activity relative to a control,indicates that the candidate compound is a compound that inhibits (is anantagonist of) HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)activity. In another embodiment, the level of biological activity of anHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide orderivative or fragment thereof in the presence of the candidate compoundto be tested, is compared with a control level that has previously beenestablished. A level of the biological activity in the presence of thecandidate compound that differs from the control level by an amount thatis statistically significant indicates that the compound alters HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) biological activity.

The present invention also relates to an assay for identifying compoundsthat alter the expression of an HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule (e.g., antisensenucleic acids, fusion proteins, polypeptides, peptidomimetics, prodrugs,receptors, binding agents, antibodies, small molecules or other drugs,or ribozymes) that alter (e.g., increase or decrease) expression (e.g.,transcription or translation) of the nucleic acid molecule or thatotherwise interact with the nucleic acids described herein, as well ascompounds identifiable by the assays. For example, a solution containinga nucleic acid encoding an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) polypeptide can be contacted with a candidate compound to betested. The solution can comprise, for example, cells containing thenucleic acid or cell lysate containing the nucleic acid; alternatively,the solution can be another solution that comprises elements necessaryfor transcription/translation of the nucleic acid. Cells not suspendedin solution can also be employed, if desired. The level and/or patternof HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) expression(e.g., the level and/or pattern of mRNA or of protein expressed, such asthe level and/or pattern of different variants) is assessed, and iscompared with the level and/or pattern of expression in a control (i.e.,the level and/or pattern of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) expression in the absence of the candidate compound, or inthe presence of the candidate compound vehicle only). If the leveland/or pattern in the presence of the candidate compound differs, by anamount or in a manner that is statistically significant, from the leveland/or pattern in the absence of the candidate compound, or in thepresence of the candidate compound vehicle only, then the candidatecompound is a compound that alters the expression of HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). Enhancement of HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) expression indicates that thecandidate compound is an agonist of HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) activity. Similarly, inhibition of HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) expression indicatesthat the candidate compound is an antagonist of HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) activity. In anotherembodiment, the level and/or pattern of an HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide(s) (e.g., different variants) inthe presence of the candidate compound to be tested, is compared with acontrol level and/or pattern that has previously been established. Alevel and/or pattern in the presence of the candidate compound thatdiffers from the control level and/or pattern by an amount or in amanner that is statistically significant indicates that the candidatecompound alters HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)expression.

In another embodiment of the invention, compounds that alter theexpression of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)nucleic acid molecule or that otherwise interact with the nucleic acidsdescribed herein, can be identified using a cell, cell lysate, orsolution containing a nucleic acid encoding the promoter region of theHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(AΔNLS) gene operablylinked to a reporter gene. After contact with a candidate compound to betested, the level of expression of the reporter gene (e.g., the level ofmRNA or of protein expressed) is assessed, and is compared with thelevel of expression in a control (i.e., the level of the expression ofthe reporter gene in the absence of the candidate compound, or in thepresence of the candidate compound vehicle only). If the level in thepresence of the candidate compound differs, by an amount or in a mannerthat is statistically significant, from the level in the absence of thecandidate compound, or in the presence of the candidate compound vehicleonly, then the candidate compound is a compound that alters theexpression of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS),as indicated by its ability to alter expression of a gene that isoperably linked to the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) gene promoter. Enhancement of the expression of the reporterindicates that the compound is an agonist of HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) activity. Similarly, inhibition of theexpression of the reporter indicates that the compound is an antagonistof HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) activity. Inanother embodiment, the level of expression of the reporter in thepresence of the candidate compound to be tested, is compared with acontrol level that has previously been established. A level in thepresence of the candidate compound that differs from the control levelby an amount or in a manner that is statistically significant indicatesthat the candidate compound alters HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) expression.

Compounds that alter the amounts of different variants encoded by HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) (e.g., a compound thatenhances activity of a first variant, and that inhibits activity of asecond variant), as well as compounds that are agonists of activity of afirst variant and antagonists of activity of a second variant, caneasily be identified using these methods described above.

In other embodiments of the invention, assays can be used to assess theimpact of a candidate compound on the activity of a polypeptide inrelation to an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)substrate, for example, an inhibitor of histone deacetylase activity.These inhibitors fall into four general classes: 1) short-chain fattyacids (e.g., 4-phenylbutyrate and valproic acid); 2) hydroxamic acids(e.g., SAHA, Pyroxamide, trichostatin A (TSA), oxamflatin and CHAPs,such as, CHAP1 and CHAP31); 3) cyclic tetrapeptides (Trapoxin A,Apicidin and Depsipeptide (FK-228, also known as FR9011228); 4)benzamides (e.g., MS-275); and other compounds such as Scriptaid.Examples of such assays and compounds can be found in U.S. Pat. No.5,369,108, issued on Nov. 29, 1994, U.S. Pat. No. 5,700,811, issued onDec. 23, 1997, and U.S. Pat. No. 5,773,474, issued on Jun. 30, 1998 toBreslow et al., U.S. Pat. No. 5,055,608, issued on Oct. 8, 1991, andU.S. Pat. No. 5,175,191, issued on Dec. 29, 1992 to Marks et al., aswell as, Yoshida et al., supra; Saito et al., supra; Furamai et al.,supra; Komatsu et al., supra; Su et al., supra; Lee et al., supra andSuzuki et al. supra, the entire content of all of which are herebyincorporated by reference.

In one example, a cell or tissue that expresses or contains a compoundthat interacts with HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) (herein referred to as an “HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate,” which can be a polypeptide orother molecule that interacts with HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS)) is contacted with HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) in the presence of a candidatecompound, and the ability of the candidate compound to alter theinteraction between HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) and the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) substrate is determined, for example, by assaying activity ofthe polypeptide. Alternatively, a cell lysate or a solution containingthe HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate,can be used. A compound that binds to HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) or the HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate can alter the interaction byinterfering with, or enhancing the ability of HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) to bind to, associate with, orotherwise interact with the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) substrate.

Determining the ability of the candidate compound to bind to HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) or an HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate can be accomplished,for example, by coupling the candidate compound with a radioisotope orenzymatic label such that binding of the candidate compound to thepolypeptide can be determined by detecting the labeled with ¹²⁵I, ³⁵S,¹⁴C, or ³H, either directly or indirectly, and the radioisotope detectedby direct counting of radioemmission or by scintillation counting.Alternatively, candidate compound can be enzymatically labeled with, forexample, horseradish peroxidase, alkaline phosphatase, or luciferase,and the enzymatic label detected by determination of conversion of anappropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a candidate compound to interact with the polypeptide without thelabeling of any of the interactants. For example, a microphysiometer canbe used to detect the interaction of a candidate compound with HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) or an HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate without the labelingof either the candidate compound, HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS), or the HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate (McConnell et al., (1992) Science,257: 1906-1912). As used herein, a “microphysiometer” (e.g.,CYTOSENSOR™) is an analytical instrument that measures the rate at whicha cell acidifies its environment using a light-addressablepotentiometric sensor (LAPS). Changes in this acidification rate can beused as an indicator of the interaction between ligand and polypeptide.

In another embodiment of the invention, assays can be used to identifypolypeptides that interact with one or more HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptides, as described herein. Forexample, a yeast two-hybrid system such as that described by Fields andSong (Fields and Song, Nature 340: 245-246 (1989)) can be used toidentify polypeptides that interact with one or more HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptides. In such a yeasttwo-hybrid system, vectors are constructed based on the flexibility of atranscription factor that has two functional domains (a DNA bindingdomain and a transcription activation domain). If the two domains areseparated but fused to two different proteins that interact with oneanother, transcriptional activation can be achieved, and transcriptionof specific markers (e.g., nutritional markers such as His and Ade, orcolor markers such as lacZ) can be used to identify the presence ofinteraction and transcriptional activation. For example, in the methodsof the invention, a first vector is used that includes a nucleic acidencoding a DNA binding domain and an HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, variant, or fragment orderivative thereof, and a second vector is used that includes a nucleicacid encoding a transcription activation domain and a nucleic acidencoding a polypeptide that potentially may interact with the HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, variant,or fragment or derivative thereof (e.g., an HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide substrate or receptor).Incubation of yeast containing the first vector and the second vectorunder appropriate conditions (e.g., mating conditions such as used inthe MATCHMAKER™ system from Clontech) allows identification of coloniesthat express the markers of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS). These colonies can be examined to identify thepolypeptide(s) that interact with the HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide or fragment or derivativethereof. Such polypeptides may be useful as compounds that alter theactivity or expression of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS),or HDRP(ΔNLS) polypeptide, as described above.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize an HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, or an HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate, or othercomponents of the assay on a solid support, in order to facilitateseparation of complexed from uncomplexed forms of one or both of thepolypeptides, as well as to accommodate automation of the assay. Bindingof a candidate compound to the polypeptide, or interaction of thepolypeptide with a substrate in the presence and absence of a candidatecompound, can be accomplished in any vessel suitable for containing thereactants. Examples of such vessels include microtitre plates, testtubes, and micro-centrifuge tubes. In one embodiment, a fusion protein(e.g., a glutathione-S-transferase fusion protein) can be provided thatadds a domain that allows HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) or an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)substrate to be bound to a matrix or other solid support.

In another embodiment, modulators of expression of nucleic acidmolecules of the invention are identified in a method wherein a cell,cell lysate, tissue, tissue lysate, or solution containing a nucleicacid encoding HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) iscontacted with a candidate compound and the expression of appropriatemRNA or polypeptide (e.g., variant(s)) in the cell, cell lysate, tissue,or tissue lysate, or solution, is determined. The level of expression ofappropriate mRNA or polypeptide(s) in the presence of the candidatecompound is compared to the level of expression of mRNA orpolypeptide(s) in the absence of the candidate compound, or in thepresence of the candidate compound vehicle only. The candidate compoundcan then be identified as a modulator of expression based on thiscomparison. For example, when expression of mRNA or polypeptide isgreater (statistically significantly greater) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator or enhancer of the mRNA or polypeptideexpression. Alternatively, when expression of the mRNA or polypeptide isless (statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of the mRNA or polypeptide expression. The level of mRNA orpolypeptide expression in the cells can be determined by methodsdescribed herein for detecting mRNA or polypeptide.

This invention further pertains to novel compounds identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use a compound identified as described hereinin an appropriate animal model. For example, a compound identified asdescribed herein (e.g., a candidate compound that is a modulatingcompound such as an antisense nucleic acid molecule, a specificantibody, or a polypeptide substrate) can be used in an animal model todetermine the efficacy, toxicity, or side effects of treatment with sucha compound. Alternatively, a compound identified as described herein canbe used in an animal model to determine the mechanism of action of sucha compound. Furthermore, this invention pertains to uses of novelcompounds identified by the above-described screening assays fortreatments as described herein. In addition, a compound identified asdescribed herein can be used to alter activity of an HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, or to alterexpression of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS),by contacting the polypeptide or the nucleic acid molecule (orcontacting a cell comprising the polypeptide or the nucleic acidmolecule) with the compound identified as described herein.

PHARMACEUTICAL COMPOSITIONS

The present invention also pertains to pharmaceutical compositionscomprising nucleic acids described herein, particularly nucleotidesencoding the polypeptides described herein; comprising polypeptidesdescribed herein (e.g., SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, and/or other variants encoded by HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)); and/or comprising a compoundthat alters (e.g., increases or decreases) HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) expression or HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide activity as described herein.For instance, a polypeptide, protein, fragment, fusion protein orprodrug thereof, or a nucleotide or nucleic acid construct (vector)comprising a nucleotide of the present invention, a compound that altersHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptideactivity, a compound that alters HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid expression, or an HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate or bindingpartner, can be formulated with a physiologically acceptable carrier orexcipient to prepare a pharmaceutical composition. The carrier andcomposition can be sterile. The formulation should suit the mode ofadministration.

Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions (e.g., NaCl), saline, buffered saline,alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzylalcohols, polyethylene glycols, gelatin, carbohydrates such as lactose,amylose or starch, dextrose, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid esters,hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well ascombinations thereof. The pharmaceutical preparations can, if desired,be mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, flavoring and/or aromatic substances andthe like that do not deleteriously react with the active compounds.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. The composition can be aliquid solution, suspension, emulsion, tablet, pill, capsule, sustainedrelease formulation, or powder. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,polyvinyl pyrollidone, sodium saccharine, cellulose, magnesiumcarbonate, etc.

Methods of introduction of these compositions include, but are notlimited to, intradermal, intramuscular, intraperitoneal, intraocular,intravenous, subcutaneous, topical, oral and intranasal. Other suitablemethods of introduction can also include gene therapy (as describedbelow), rechargeable or biodegradable devices, particle accelerationdevises (“gene guns”) and slow release polymeric devices. Thepharmaceutical compositions of this invention can also be administeredas part of a combinatorial therapy with other compounds.

The composition can be formulated in accordance with the routineprocedures as a pharmaceutical composition adapted for administration tohuman beings. For example, compositions for intravenous administrationtypically are solutions in sterile isotonic aqueous buffer. Wherenecessary, the composition may also include a solubilizing agent and alocal anesthetic to ease pain at the site of the injection. Generally,the ingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampule orsachette indicating the quantity of active compound. Where thecomposition is to be administered by infusion, it can be dispensed withan infusion bottle containing sterile pharmaceutical grade water, salineor dextrose/water. Where the composition is administered by injection,an ampule of sterile water for injection or saline can be provided sothat the ingredients may be mixed prior to administration.

For topical application, nonsprayable forms, viscous to semi-solid orsolid forms comprising a carrier compatible with topical application andhaving a dynamic viscosity preferably greater than water, can beemployed. Suitable formulations include but are not limited tosolutions, suspensions, emulsions, creams, ointments, powders, enemas,lotions, sols, liniments, salves, aerosols, etc., that are, if desired,sterilized or mixed with auxiliary agents, e.g., preservatives,stabilizers, wetting agents, buffers or salts for influencing osmoticpressure, etc. The compound may be incorporated into a cosmeticformulation. For topical application, also suitable are sprayableaerosol preparations wherein the active ingredient, preferably incombination with a solid or liquid inert carrier material, is packagedin a squeeze bottle or in admixture with a pressurized volatile,normally gaseous propellant, e.g., pressurized air.

Compounds described herein can be formulated as neutral or salt forms.Pharmaceutically acceptable salts include those formed with free aminogroups such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with free carboxyl groupssuch as those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The compounds are administered in a therapeutically effective amount.The amount of compounds that will be therapeutically effective in thetreatment of a particular disorder or condition will depend on thenature of the disorder or condition, and can be determined by standardclinical techniques. In addition, in vitro or in vivo assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the symptoms of a cellproliferation disease, an apoptotic disease, or a cell differentiationdisease, and should be decided according to the judgment of apractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, that notice reflects approval bythe agency of manufacture, use of sale for human administration. Thepack or kit can be labeled with information regarding mode ofadministration, sequence of drug administration (e.g., separately,sequentially or concurrently), or the like. The pack or kit may alsoinclude means for reminding the patient to take the therapy. The pack orkit can be a single unit dosage of the combination therapy or it can bea plurality of unit dosages. In particular, the compounds can beseparated, mixed together in any combination, present in a single vialor tablet. Compounds assembled in a blister pack or other dispensingmeans is preferred. For the purpose of this invention, unit dosage isintended to mean a dosage that is dependent on the individualpharmacodynamics of each compound and administered in FDA approveddosages in standard time courses.

METHODS OF THERAPY

The present invention also pertains to methods of treatment(prophylactic, diagnostic, and/or therapeutic) for a cell proliferationdisease, an apoptotic disease, or a cell differentiation disease, usingan HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) therapeuticcompound. An “HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)therapeutic compound” is a compound that alters (e.g., enhances orinhibits) HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)polypeptide activity and/or HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) nucleic acid molecule expression, as described herein (e.g.,an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) agonist orantagonist). HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)therapeutic compounds can alter HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide activity or nucleic acidmolecule expression by a variety of means, such as, for example, byproviding additional HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) polypeptide or by upregulating the transcription ortranslation of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) nucleic acid molecule; by altering post-translationalprocessing of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) polypeptide; by altering transcription of HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) variants; or by interferingwith HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptideactivity (e.g., by binding to an HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide), or by downregulating thetranscription or translation of the HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule. Representative HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) therapeutic compoundsinclude the following: nucleic acids or fragments or derivatives thereofdescribed herein, particularly nucleotides encoding the polypeptidesdescribed herein and vectors comprising such nucleic acids (e.g., anucleic acid molecule, cDNA, and/or RNA, such as a nucleic acid encodingan HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptideor active fragment or derivative thereof, or an oligonucleotide; forexample, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQID NO: 9, which may optionally comprise at least one polymorphism, or anucleic acid encoding SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, or fragments or derivatives thereof); polypeptidesdescribed herein (e.g., SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8 SEQ ID NO: 10 and/or other variants encoded by HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), or fragments or derivativesthereof); HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)substrates; peptidomimetics; fusion proteins or prodrugs thereof;antibodies (e.g., an antibody to a mutant HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, or an antibody to a non-mutantHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, oran antibody to a particular variant encoded by HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), as described above);ribozymes; other small molecules; and other compounds that alter (e.g.,enhance or inhibit) HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) nucleic acid expression or polypeptide activity, for example,those compounds identified in the screening methods described herein, orthat regulate transcription of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS),or HDRP(ΔNLS) variants (e.g., compounds that affect which variants areexpressed, or that affect the amount of each variant that is expressed.More than one HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)therapeutic compound can be used concurrently, if desired.

The HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) therapeuticcompound that is a nucleic acid is used in the treatment of a cellproliferation disease, an apoptotic disease, or a cell differentiationdisease. The term, “treatment” as used herein, refers not only toameliorating symptoms associated with the disease, but also preventingor delaying the onset of the disease, and also lessening the severity orfrequency of symptoms of the disease. The therapy is designed to alter(e.g., inhibit or enhance), replace or supplement activity of an HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide in anindividual. For example, an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) therapeutic compound can be administered in order toupregulate or increase the expression or availability of the HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid moleculeor of specific variants of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS), or, conversely, to downregulate or decrease the expressionor availability of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) nucleic acid molecule or specific variants of HDAC9, H{DAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). Upregulation or increasingexpression or availability of a native HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule or of a particularvariant could interfere with or compensate for the expression oractivity of a defective gene or another variant; downregulation ordecreasing expression or availability of a native HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule or of aparticular variant could minimize the expression or activity of adefective gene or the particular variant and thereby minimize the impactof the defective gene or the particular variant.

The HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) therapeuticcompound(s) are administered in a therapeutically effective amount(i.e., an amount that is sufficient to treat the disease, such as byameliorating symptoms associated with the disease, preventing ordelaying the onset of the disease, and/or also lessening the severity orfrequency of symptoms of the disease). The amount that will betherapeutically effective in the treatment of a particular individual'sdisorder or condition will depend on the symptoms and severity of thedisease, and can be determined by standard clinical techniques. Inaddition, in vitro or in vivo assays may optionally be employed to helpidentify optimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of a practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

In one embodiment, a nucleic acid of the invention (e.g., a nucleic acidencoding an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)polypeptide, such as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, or SEQ ID NO: 9, which may optionally comprise at least onepolymorphism, or a nucleic acid that encodes an HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide or a variant,derivative or fragment thereof, such as a nucleic acid encoding theprotein of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, orSEQ ID NO: 10) can be used, either alone or in a pharmaceuticalcomposition as described above. For example, HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) or a cDNA encoding an HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, either by itselfor included within a vector, can be introduced into cells (either invitro or in vivo) such that the cells produce native HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide. If desired, cellsthat have been transformed with the gene or cDNA or a vector comprisingthe gene or cDNA can be introduced (or re-introduced) into an individualaffected with the disease. Thus, cells that, in nature, lack nativeHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) expression andactivity, or have mutant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) expression and activity, or have expression of adisease-associated HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) variant, can be engineered to express an HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide or an activefragment of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)polypeptide (or a different variant of an HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide). In a preferred embodiment,nucleic acid encoding the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) polypeptide, or an active fragment or derivative thereof, canbe introduced into an expression vector, such as a viral vector, and thevector can be introduced into appropriate cells in an animal. Other genetransfer systems, including viral and nonviral transfer systems, can beused. Alternatively, nonviral gene transfer methods, such as calciumphosphate coprecipitation, mechanical techniques (e.g., microinjection);membrane fusion-mediated transfer via liposomes; or direct DNA uptake,can also be used to introduce the desired nucleic acid molecule into acell.

Alternatively, in another embodiment of the invention, a nucleic acid ofthe invention; a nucleic acid complementary to a nucleic acid of theinvention; or a portion of such a nucleic acid (e.g., an oligonucleotideas described below), can be used in “antisense” therapy, in which anucleic acid (e.g., an oligonucleotide) that specifically hybridizes tothe RNA and/or genomic DNA of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS),or HDRP(ΔNLS) is administered or generated in situ. The antisensenucleic acid that specifically hybridizes to the RNA and/or DNA inhibitsexpression of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) nucleic acid molecule, e.g., by inhibiting translation and/ortranscription. Binding of the antisense nucleic acid can be byconventional base pair complementarity, or, for example, in the case ofbinding to DNA duplexes, through specific interaction in the majorgroove of the double helix.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid as described above. When the plasmidis transcribed in the cell, it produces RNA that is complementary to aportion of the mRNA and/or DNA that encodes an HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide. Alternatively, theantisense construct can be an oligonucleotide probe which is generatedex vivo and introduced into cells; it then inhibits expression byhybridizing with the mRNA and/or genomic DNA of HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). In one embodiment, theoligonucleotide probes are modified oligonucleotides that are resistantto endogenous nucleases, e.g. exonucleases and/or endonucleases, therebyrendering them stable in vivo. Exemplary nucleic acid molecules for useas antisense oligonucleotides are phosphoramidate, phosphothioate andmethylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996;5,264,564; and 5,256,775). Additionally, general approaches toconstructing oligomers useful in antisense therapy are also described,for example, by Van der Krol et al., Biotechniques 6: 958-976 (1988);and Stein et al., Cancer Res 48: 2659-2668 (1988). With respect toantisense DNA, oligodeoxyribonucleotides derived from the translationinitiation site, e.g. between the −10 and +10 regions of an HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid sequence,are preferred.

To perform antisense therapy, oligonucleotides (RNA, cDNA or DNA) aredesigned that are complementary to mRNA encoding an HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide. The antisenseoligonucleotides bind to HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) mRNA transcripts and prevent translation. Absolutecomplementarity, although preferred, is not required. A sequence“complementary” to a portion of an RNA, as referred to herein, indicatesthat a sequence has sufficient complementarity to be able to hybridizewith the RNA, forming a stable duplex; in the case of double-strandedantisense nucleic acids, a single strand of the duplex DNA may thus betested, or triplex formation may be assayed. The ability to hybridizewill depend on both the degree of complementarity and the length of theantisense nucleic acid, as described in detail above. Generally, thelonger the hybridizing nucleic acid, the more base mismatches with anRNA it may contain and still form a stable duplex (or triplex, as thecase may be). One skilled in the art can ascertain a tolerable degree ofmismatch by use of standard procedures.

The oligonucleotides used in antisense therapy can be DNA, RNA, orchimeric mixtures or derivatives or modified versions thereof,single-stranded or double-stranded. The oligonucleotides can be modifiedat the base moiety, sugar moiety, or phosphate backbone, for example, toimprove stability of the molecule, hybridization, etc. Theoligonucleotides can include other appended groups such as peptides(e.g. for targeting host cell receptors in vivo), or compoundsfacilitating transport across the cell membrane (see, e.g., Letsinger etal., Proc. Natl. Acad. Sci. USA 86: 6553-6556 (1989); Lemaitre et al.,Proc. Natl. Acad Sci. USA 84: 648-652 (1987); PCT InternationalPublication No. W088/09810)) or the blood-brain barrier (see, e.g., PCTInternational Publication No. W089/10134), or hybridization-triggeredcleavage agents (see, e.g., Krol et al., BioTechniques 6: 958-976(1988)) or intercalating agents. (See, e.g., Zon, Pharmn. Res. 5:539-549 (1988)). To this end, the oligonucleotide may be conjugated toanother molecule (e.g., a peptide, hybridization triggered cross-linkingagent, transport agent, hybridization-triggered cleavage agent).

The antisense molecules are delivered to cells that express HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) in vivo. A number ofmethods can be used for delivering antisense DNA or RNA to cells; e.g.,antisense molecules can be injected directly into the tissue site, ormodified antisense molecules, designed to target the desired cells(e.g., antisense linked to peptides or antibodies that specifically bindreceptors or antigens expressed on the target cell surface) can beadministered systematically. Alternatively, in a preferred embodiment, arecombinant DNA construct is utilized in which the antisenseoligonucleotide is placed under the control of a strong promoter (e.g.,pol III or pol II). The use of such a construct to transfect targetcells in the patient results in the transcription of sufficient amountsof single stranded RNAs that will form complementary base pairs with theendogenous HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)transcripts and thereby prevent translation of the HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) mRNA. For example, a vector canbe introduced in vivo such that it is taken up by a cell and directs thetranscription of an antisense RNA. Such a vector can remain episomal orbecome chromosomally integrated, as long as it can be transcribed toproduce the desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art and describedabove. For example, a plasmid, cosmid, YAC, or viral vector can be usedto prepare the recombinant DNA construct that can be introduced directlyinto the tissue site. Alternatively, viral vectors can be used thatselectively infect the desired tissue, in which case administration maybe accomplished by another route (e.g., systematically).

Endogenous HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)expression can also be reduced by inactivating or “knocking out” HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid sequencesor their promoters using targeted homologous recombination (e.g., seeSmithies et al., Nature 317: 230-234 (1985); Thomas and Capecchi, Cell51: 503-512 (1987); Thompson et al., Cell 5: 313-321 (1989)). Forexample, a mutant, non-functional HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) (or a completely unrelated DNA sequence)flanked by DNA homologous to the endogenous HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) (either the coding regions or regulatoryregions of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)) canbe used, with or without a selectable marker and/or a negativeselectable marker, to transfect cells that express HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) in vivo. Insertion of the DNAconstruct, via targeted homologous recombination, results ininactivation of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS).The recombinant DNA constructs can be directly administered or targetedto the required site in vivo using appropriate vectors, as describedabove. Alternatively, expression of non-mutant HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) can be increased using asimilar method: Targeted homologous recombination can be used to inserta DNA construct comprising a non-mutant, functional HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) (e.g., a gene having SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, which mayoptionally comprise at least one polymorphism), or a portion thereof, inplace of a mutant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) in the cell, as described above. In another embodiment,targeted homologous recombination can be used to insert a DNA constructcomprising a nucleic acid that encodes an HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide variant that differs from thatpresent in the cell.

Alternatively, endogenous HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) expression can be reduced by targeting deoxyribonucleotidesequences complementary to the regulatory region of HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) (i.e., the HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) promoter and/or enhancers) toform triple helical structures that prevent transcription of HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) in target cells in thebody. (See generally, Helene Anticancer Drug Des., 6(6): 569-84(1991);Helene et al., Ann, N.Y. Acad. Sci., 660: 27-36 (1992); and Maher,Bioassays 14(12): 807-15 (1992)). Likewise, the antisense constructsdescribed herein, by antagonizing the normal biological activity of oneof the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) proteins,can be used in the manipulation of tissue, e.g., tissue differentiation,both in vivo and for ex vivo tissue cultures. Furthermore, the antisensetechniques (e.g., microinjection of antisense molecules, or transfectionwith plasmids whose transcripts are anti-sense with regard to an HDAC9,HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) mRNA or gene sequence)can be used to investigate role of HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) in developmental events, as well as thenormal cellular function of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), orHDRP(ΔNLS) in adult tissue. Such techniques can be utilized in cellculture, but can also be used in the creation of transgenic animals.

In yet another embodiment of the invention, other HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) therapeutic compounds asdescribed herein can also be used in the treatment or prevention of acell proliferation disease, an apoptotic disease, or a celldifferentiation disease. The therapeutic compounds can be delivered in acomposition, as described above, or by themselves. They can beadministered systemically, or can be targeted to a particular tissue.The therapeutic compounds can be produced by a variety of means,including chemical synthesis; recombinant production; in vivo production(e.g., a transgenic animal, such as U.S. Pat. No. 4,873,316 to Meade etal.), for example, and can be isolated using standard means such asthose described herein.

A combination of any of the above methods of treatment (e.g.,administration of non-mutant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS),or HDRP(ΔNLS) polypeptide in conjunction with antisense therapytargeting mutant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)mRNA; administration of a first variant encoded by HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) in conjunction with antisensetherapy targeting a second encoded by HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS), can also be used.

In another embodiment, the invention is directed to HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecules andHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptides foruse as a medicament in therapy. For example, the nucleic acid moleculesor polypeptides of the present invention can be used in the treatment ofa cell proliferation disease, an apoptotic disease, or a celldifferentiation disease. In addition, the HDAC9, HDAC9a, HDAC9(ΔNLS),HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecules and HDAC9, HDAC9a,HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptides described hereincan be used in the manufacture of a medicament for the treatment of acell proliferation disease, an apoptotic disease, or a celldifferentiation disease.

The invention will be further described by the following non-limitingexamples. The teachings of all publications cited herein areincorporated herein by reference in their entirety.

EXEMPLIFICATION

Cloning of cDNA Encodes a Novel HDAC, Designated HDAC9

HDAC9 was cloned by PCR and 3′ rapid amplification of cDNA ends usingprimers designed from the sequence of human chromosome 7 whosetranslated product exhibited 80% identity to the HDAC domain of HDAC4,described in detail as follows.

Database analyses indicate that HDRP is located on chromosome 7(7p15-p21). The human genome database (February 2001 release) of GenBankwas searched using the human HDAC4 amino acid sequence. The TBLASTNprogram was used to identify open reading frames downstream of HDRP onchromosome 7 that exhibit significant homology to the HDAC domain ofHDAC4. Several fragments whose translated products exhibit over 58%identity were retrieved. Two sense primers (OL486,5′-CCATGGAAACGGTACCCAGCAGGC-3′ (SEQ ID NO: 16) and OL487,5′-CACTCCATCGCTATGATGAAGGG-3′ (SEQ ID NO: 17)) and antisense primers(OL484, 5′-AGTTCCCTTCATCATAGCGATGG-3′ (SEQ ID NO: 18) and OL485,5′-AATGTACAGGATGCTGGGGT-3′ (SEQ ID NO: 19)) each were designed basedupon one of these fragments whose translated products matched aminoacids 842-873 of HDAC4. RT-PCR was performed using each of the antisenseprimers and a sense primer (5′-CCCTTGTAGCTGGTGGAGTTCCCTT-3′ (SEQ ID NO:20)) from the coding region of HDRP and human brain cDNA as a template.PCR was performed in a Biometra TGRADIENT Thermocycler for 30 cycles at95° C. for 20 seconds, 60° C. for 20 seconds, and 72° C. for 120seconds.

3′-rapid amplification of cDNA ends was performed using the sense primerOL486 and adaptor primer 1 (Clontech), and marathon-ready cDNA fromhuman brain (Clontech, Palo Alto, Calif.) according to themanufacturer's instruction. The products were re-amplified using nestedsense primer OL487 and adaptor primer 2 (Clontech, Palo Alto, Calif.).PCR products were cloned into pGEM-T-easy vector (Promega, Madison,Wis.) and sequenced using an automated DNA sequencer at the DNASequencing Core Facility of the Memorial Sloan-Kettering Cancer Center,using DNA sequencing methods known to one of skill in the art.

Two cDNAs were cloned from the above-described methods. One cDNA (SEQ IDNO: 1) encodes-an HDAC9 protein that is 1011 amino acids in length. Theother cDNA (SEQ IID NO: 3) encodes an HDAC9a protein that is 879 aminoacids long. The cDNA sequence and amino sequence of HDAC9 and HDAC9a areshown in FIGS. 1A-1G and FIGS. 2A-2B, respectively. Database analyses ofthese cDNAs against human genomic DNA sequences indicated that these twocDNAs are generated by alternatively splicing. An alignment of HDAC9,HDAC9a, HDRP, and HDAC4 is shown in FIGS. 3A-3C.

Each of the HDAC9 and HDAC9a nucleic acid sequences were cloned into thepFLAG-CMV-5b vector (Sigma) in frame with the C-terminal FLAG tag. Onlythe coding regions plus three extra base pairs (ACC) of cDNA of theHDAC9 and HDAC9a nucleic acid sequences were included in the constructs.These constructs are referred to herein as HDAC9-FLAG and HDAC9a-FLAG,respectively. These constructs are contained in E. coli, and can readilybe expressed. For HDAC9, the insert is 3033 bp and for HDAC9a, theinsert size is 2637 bp. Both HDAC9 and HDAC9a can be released with EcoRVand BamHI (whose sites have been incorporated in the primers to obtainHDAC9 and HDAC9a coding cDNA for cloning purpose) restriction enzymedigestion.

The HDAC9 cDNA sequences from the known 5′-end of HDRP cDNA to the3′-untranslated region cloned in this study cover over 511 kb of genomicDNA on chromosome 7. As shown in FIG. 4, the coding region cDNA of HDAC9resides in 23 exons spanning 458 kb of genomic sequence. Exons 21, 22,and 23 are one single exon in HDAC9a, but the middle exon that isnumbered exon 22 in FIG. 4, containing an in-frame stop codon, isspliced out in HDAC9. In addition, exons 12 and 13 are a single exonused by HDRP. Exon 13 is spliced as part of an intron in HDAC9 andHDAC9a.

Further analysis revealed that exon 7, which contains a nuclearlocalization signal (NLS) is alternatively spliced in an HDRP isoform,creating HDRP(ΔNLS). RT-PCR analyses using primers based on sequencesfrom exon 6 and exon 14 indicate that this alternative splicing eventalso occurs in HDAC9 and/or HDAC9a. Thus, it is possible that at least 6proteins can be generated from a single HDAC9 gene by alternativelysplicing of its RNA. The cDNA sequences and amino acid sequences forHDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) are shown inFIGS. 1A-1O and 2A-2E, respectively. HDAC9 mRNA is differentiallyexpressed among human tissues The expression of HDAC9 mRNA wasdetermined by Northern blot analysis using a human multiple tissueNorthern blot (Clontech, Palo Alto, Calif.). Hybridization was performedaccording to the manufacturer's instruction using ExPressHyb solution(Clontech, Palo Alto, Calif.). The ³²P-random priming labeled3′-untranslated region common to both HDAC9 and HDAC9a that shares nosignificant sequence homology with HDRP was used as a probe. Twotranscripts at 9.8 and 4.1 kb were detected in all tissues examined(FIG. 6A). The 4.1 kb transcript is shorter than the 4.4 kb HDRPtranscript (See Zhou, et al., Proc. Natl. Acad. Sci. USA, 97: 1056-1061(2000)). A third transcript at 1.2 kb was detected in placenta (FIG.6A). Similar to HDRP (See Zhou, X., et al., Proc. Natl. Acad. Sci. USA,97: 1056-1061 (2000)), high levels of HDAC9 transcripts were detected inbrain and skeletal muscle (FIG. 6A).

The distribution of alternatively spliced mRNA variants among tissueswas examined by RT-PCR using primers (OL516 5′-TGTGTCATCGAGCTGGCTTC-3′(SEQ ID NO: 21) and OL517 5′-ATCTTCTGCAAGTGGCTCCA-3′ (SEQ ID NO: 22))spanning the alternatively spliced exon 22 and cDNA panel from the sametissues as the multiple tissue Northern blot. PCR was performed in aBiometra TGRADIENT Thermocycler for 30 cycles at 95° C. for 20 seconds,60° C. for 20 seconds, and 72° C. for 60 seconds. The expected sizes ofPCR products were 680 base pairs for HDAC9 and 993 base pairs forHDAC9a. The ratio of HDAC9 and HDAC9a transcripts differed among tissues(FIG. 6B). In the placenta and kidney, the levels of the two transcriptswere about the same (FIG. 6B). In the brain, heart, and pancreas, therewere more transcripts of HDAC9 than HDAC9a. In the other tissuesexamined, there were more HDAC9a transcripts than HDAC9 transcripts(FIG. 6B). Under the conditions tested, HDAC9 transcripts wereundetectable in liver (FIG. 6B). The lung had an HDAC9 product that waslarger than expected and abundant. The lung also had low levels of HDAC9transcripts and HDAC9a transcripts (FIG. 6B). An additional PCR productwas also amplified from cDNA of the pancreas; this product was than theexpected products from HDAC9 and HDAC9a (FIG. 6B). The identity of thedifferent sized transcripts is unknown.

HDAC9 and HDAC9a Possess Histone Deacetylase Activity

HDAC9 was named based on sequence homology to HDAC4 (FIGS. 3A-3C). Todetermine whether HDAC9 and HDAC9a possess HDAC activity, an HDACenzymatic assay was performed using anti-FLAG immunoprecipitatedHDAC9-FLAG and HDAC9a-FLAG.

C-terminal FLAG-tagged HDAC9 (HDAC9-FLAG) and HDAC9a (HDAC9a-FLAG)expression vectors were constructed using the pFLAG-CMV-5b vector(Sigma) and PCR amplified coding regions of HDAC9 and HDAC9a in framewith the FLAG-tag to form pFLAG-CMV-5b-HDAC9 (plasmid VR1) andpFLAG-CMV-5b-HDAC9a (plasmid VR2). All constructs were confirmed by DNAsequencing.

Transfection of human kidney 293T cells, immunoprecipitation usinganti-FLAG M2 Agarose (Sigma), Western blot analyses and dual luciferaseassays were performed essentially as previously described by Zhou et al.(Proc. Natl. Acad. Sci. USA, 97: 1056-1061 (2000)). Briefly, the cells(American Type Culture Collection) were cultured in DME HG medium(GIBCO/BRL) supplemented with 10% (vol/vol) FBS at 37° C. in a 5% CO₂atmosphere. Transient transfection was performed by using Lipofectamine(GIBCO/BRL) or Fugene 6 (Roche Molecular Biochemicals) according to themanufacturers' instructions. Cells were harvested 24 to 48 hours aftertransfection and lysed in IP lysis buffer (50 mM Tris·HCl, pH 7.5/120 mMNaCl/5 mM EDTA/0.5% NP-40) at 5×107 cells per ml. Immunoprecipitationwith anti-FLAG M2-agarose (Sigma, St. Louis, Miss.) was performedaccording to the manufacturer's instructions. Immunoprecipitatedproteins were released from the agarose beads by using FLAG-peptide andeither used directly for HDAC enzymatic activity assays or resolved onSDS/PAGE for Western blot analyses. Anti-FLAG antibody was purchasedfrom Sigma (St. Louis, Miss.). Western blot analyses were performedusing standard methods.

HDAC9 and HDAC9a enzymatic activity were assessed with the HDACFluorescent Activity Assay/Drug Discovery Kit-AK-500 (BIOMOL ResearchLaboratories) using a FLUOR DE LYS™ that contains an acetylated lysineside chain as a substrate and immunoprecipitated HDAC9-FLAG andHDAC9a-FLAG polypeptides according to the manufacturer's instruction anda SPECTRAmax® GEMINI XS microplate spectrofluorometer using the SOFTmax®PRO system (Molecular Devices) at excitation 355 nm and emission 460 nmwith a cut off filter of 455 nm. Briefly, HDAC9-FLAG and HDAC9a-FLAGwere incubated with the substrate overnight at room temperature in a96-well plate. The reaction was stopped by addition of Fluor De Lys™Developer and samples were read with the fluorometer.

As shown in FIG. 7, both HDAC9-FLAG and HDAC9a-FLAG deacetylated theacetylated lysine of FLUOR DE LYS™ and the activity of HDAC9 and HDAC9awas comparable. To examine the activity of HDAC9 and HDAC9a, inhibitionstudies using TSA were carried out by preincubating HDAC9-FLAG andHDAC9a-FLAG with TSA for 15 minutes at room temperature. The assay wasthen carried out as stated above. As shown in FIG. 7, TSA inhibitedHDAC9 and HDAC9a deacetylase activity. The inset gel in FIG. 7 shows theamount of protein used in the assay. SAHA, a potent HDAC inhibitor(Richon et al., Proc. Natl. Acad. Sci. USA, 95: 3003-3007 (1998)) alsocompletely inhibited the histone deacetylase activity of HDAC9-FLAG andHDAC9a-FLAG. The HDAC activity of HDAC9 and HDAC9a was about ten timeslower than the deacetylase activity of HDAC4 when comparable amount ofprotein was used under conditions tested here.

HDAC9 and HDAC9a enzymatic activity was also determined through HDACenzymatic assays using ³H-histones isolated from murine erythroleukeriacells as a substrate. This assay was performed essentially as describedby Richon et al. (Proc. Natl. Acad. Sci. USA, 95: 3003-3007 (1998)).Briefly, HDAC9-FLAG and HDAC9a-FLAG were incubated with ³H-histonesovernight at 37° C. The reaction was stopped by the addition of 1MHCl/0.1 acetic acid. Released ³H-acetic acid was extracted with ethylacetate and quantified by scintillation counting. For inhibitionstudies, the immunoprecipitated complexes were preincubated with thedifferent HDAC inhibitors for 30 minutes at 4° C.

As shown in FIG. 8, HDAC9a-FLAG deacetylated ³H-acetyl-histones. SAHA, apotent HDAC inhibitor also completely inhibited the histone deacetylaseactivity of HDAC9a-FLAG. TSA also inhibited HDAC9a deacetylase activity.Similar results were obtained when HDAC9 was used as the enzyme source.

HDAC9 and HDAC9a Repress MEF2-Mediated Transcription

The Xenopus homolog of HDRP, MITR, was identified as a MEF2 interactingtranscriptional repressor (Sparrow et al., EMBO J. 18: 5085-5098(1999))and mouse HDRP also interacts with and represses MEF2 mediatedtranscription (Zhang et al., J. Biol. Chem. 276: 35-39 (2001)). We firsttested whether HDAC9-FLAG and HDAC9a-FLAG interact with MF2. 293 cellswere transfected with vector, HDAC9-FLAG, or HDAC9a-FLAG. The cells weresubsequently lysed and HDAC9-FLAG and HDAC9a-FLAG proteins wereimmunoprecipitated with anti-FLAG antibodies. Western blot analysis ofthe immunoprecipitated proteins was carried out, using anti-MEF-2antibody to probe the blot. As shown in FIG. 9A, both HDAC9 and HDAC9ainteracted with MEF2 in 293T cells.

It was then determined whether HDAC9 and HDAC9a repress MEF2-mediatedtranscription. This determination was carried out as follows. Thep3XMEF2-luciferase reporter gene (100 ng) and the vector pRL-TK(Promega) (5 ng) were co-transfected into 293T cells in the absence(pcDNA3 empty vector) or presence of MEF2C (100 ng of pCMV-MEF2C).HDAC9-F (1 ng, 10 ng, or 100 ng of pFLAG-HDAC9; pFLAG-HDAC9 andHDAC9-FLAG are different constructs, with the FLAG sequence located atopposite ends of the HDAC9 nucleotide, but are functionally equivalent)or HDAC9a-F (1 ng, 10 ng, or 100 ng of pFLAG-HDAC9a; pFLAG-HDAC9a andHDAC9a-FLAG are different constructs, with the FLAG sequence located atopposite ends of the HDAC9a nucleotide, but are functionally equivalent)was included in a subset of experimental groups with the MEF2C vector.pFLAG empty vector was used to adjust the DNA to an equal amount in eachtransfection. The cells were harvested 24 to 36 hours after transfectionand the luciferase activities were measured using the Dual-LuciferaseReporter Assay System from Promega according to the manufacturer'sinstruction. The firefly luciferase activity was first normalized to theco-transfected Renilla luciferase activity (encoded by the pRL-TKvector), and the luciferase activity-value for cells transfected withMEF2C alone was set at 1. MEF2C activated transcription over 30 timesthe basal level of transcription. As shown in FIG. 9B, HDAC9-FLAG andHDAC9a-FLAG repressed MEF2C mediated transcriptional activation in adose-dependent manner and completely abolished the activation at the 100ng dose for both HDAC9 and HDAC9a. The transcriptional repression effectof HDAC9 and HDAC9a on MEF2C mediated transcription was a specificeffect since a co-transfected reporter gene for transfection efficiencycontaining a TK promoter was not repressed by HDAC9 or HDAC9a.

Described herein is the identification and characterization of a newclass II HDAC, designated HDAC9. HDAC9 has several alternatively splicedisoforms, one of which is the previously identified HDRP (Zhou et al.,Proc. Natl. Acad. Sci. USA 97: 1056-1061 (2000)). HDAC9 and HDAC9apossess HDAC activity, which appears to have a lower specific enzymaticactivity than HDAC4. While not wishing to be bound by any particulartheory, it is possible that an essential co-factor is lost duringimmunoprecipitation or does not exist in 293T cells (for example,metastasis-associated protein 2 is essential for the assembly of acatalytically active HDAC1 (Zhang et al., Genes Dev. 13: 1924-1935(1999)), the substrates used are not its natural substrate, or the FLAGtag which interferes with the folding of the protein.

Searching the human genome with the HDAC domain from either HDAC1 orHDAC9 identified a total of 10 HDACs in the presently completed humangenome sequence, a number of which are schematically represented in FIG.10. HDACs 1, 2, 3, 8, 4, 5, 6, 7, 9, and 9a all have HDAC domains. HDRP,which is also schematically depicted m FIG. 10, does not have acatalytic domain.

All references described herein are incorporated by reference in theirentirety. While this invention has been particularly shown and describedwith reference to preferred embodiment thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

1-3. (canceled)
 4. An isolated nucleic acid molecule selected from thegroup: a) an isolated nucleic acid comprising SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; b) a complement of anisolated nucleic acid comprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO: 7, or SEQ ID NO: 9; c) an isolated nucleic acid encoding ahistone deacetylase polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, or SEQ ID NO: 10; d) a complement of an isolatednucleic acid encoding a histone deacetylase polypeptide of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10; e) a nucleicacid that is hybridizeable under high stringency conditions to a nucleicacid molecule that encodes any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, or SEQ ID NO: 8, or a complement thereof; or f) a nucleic acidmolecule that is hybridizeable under high stringency conditions to anucleic acid comprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQID NO: 7; and g) an isolated nucleic acid molecule that has at least 55%sequence identity with any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 9, or a complement thereof.
 5. The isolatednucleic acid molecule of claim 4, said nucleic acid molecule consistingof the nucleic acid molecule selected from the group consisting of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO:
 9. 6.The isolated nucleic acid molecule of claim 4, wherein said nucleic acidmolecule is human.
 7. A vector comprising the isolated nucleic acidmolecule of claim
 4. 8. A cell comprising the vector of claim
 7. 9. Acell comprising the isolated nucleic acid molecule of claim
 4. 10.(canceled)
 11. A method of identifying a compound that modulatesexpression of a nucleic acid molecule of claim 4, said method comprisingthe steps of: a) contacting said nucleic acid molecule with a candidatecompound under conditions suitable for expression; and b) assessing thelevel of expression of said nucleic acid molecule, wherein a candidatecompound that increases or decreases expression of said nucleic acidmolecule relative to a control is a compound that modulates expressionof said nucleic acid molecule.
 12. The method of claim 11, wherein saidmethod is carried out in a cell or animal.
 13. The method of claim 11,wherein said method is carried out in a cell free system. 14-25.(canceled)
 26. A method of identifying a compound that modulatesexpression of a nucleic acid molecule of claim 4, said method comprisingthe steps of: a) providing a nucleic acid molecule comprising a promoterregion of said nucleic acid of claim 4 or part of a promoter region ofsaid nucleic acid of claim 4 operably linked to a reporter gene; b)contacting said nucleic acid molecule or with a candidate compound; andc) assessing the level of said reporter gene, wherein a candidatecompound that increases or decreases expression of said reporter generelative to a control is a compound that modulates expression of saidnucleic acid molecule of claim
 4. 27. The method of claim 26, whereinsaid method is carried out in a cell. 28-31. (canceled)
 32. A method ofdiagnosing a cell proliferation disease, an apoptotic disease, or a celldifferentiation disease in a subject, said method comprising the stepsof: a) obtaining a sample from said subject; and b) detecting the levelof said nucleic acid molecule of claim 4; wherein if said level isincreased relative to a control, then said subject has an increasedlikelihood of having a cell proliferation disease, an apoptotic disease,or a cell differentiation disease, and wherein if said level isdecreased relative to a control, then said subject has a decreasedlikelihood of having a cell proliferation disease, an apoptotic disease,or a cell differentiation disease.
 33. The method of claim 32, whereinsaid level of said nucleic acid molecule of claim 4 in said sample ismeasured using in situ hybridization techniques. 34-35. (canceled)